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vii-«vs Michigan State
Z University
20:6

This is to certify that the
thesis entitled

NEW INSIGHTS IN THE UREASE ACTIVATION PROCESS
OBTAINED BY CHARACTERIZATION OF APOUREASE
COMPLEXES AND THE UreG ACCESSORY PROTEIN OF
KLEBSIELLA AEROGENES

presented by

Soledad De Los Angeles Quiroz Valenzuela

has been accepted towards fulfillment
of the requirements for the

Doctoral degree in Biochemistry and Molecular
Biology

 

 

 

Major Professor’s gfgnature

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v

 

Date

MSU is an affinnative-action, equal-opportunity employer

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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5I08 K lProi/Acc8Pres/CIRC/Date0ue Indd

NEW INSIGHTS IN THE UREASE ACTIVATION PROCESS OBTAINED BY
CHARACTERIZATION OF APOUREASE COMPLEXES AND THE UreG
ACCESSORY PROTEIN OF KLEBSIELLA AEROGENES
By

Soledad De Los Angeles Quiroz Valenzuela

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHiLOSOPHY
Department of Biochemistry and Molecular Biology

2008

ABSTRACT
NEW INSIGHTS IN THE UREASE ACTIVATION PROCESS OBTAINED BY
CHARACTERIZATION OF APOUREASE COMPLEXES AND THE UreG
ACCESSORY PROTEIN OF KLEBSIELLA AEROGENES
BY

Soledad De Los Angeles Quiroz Valenzuela

The metallocenter assembly pathway of nickel-containing urease from Klebsiella
aerogenes has served as a paradigm for understanding the biosynthesis of other
metalloproteins. Our current understanding of the process is that urease
apoprotein (UreABC)3 interacts with a urease-specific molecular Chaperone
(UreDFG) and a metallochaperone (UreE) that delivers nickel ions. At each of the
three active sites, a specific lysine residue is carbamylated and dinuclear nickel
centers are formed in a GTP-dependent process. Urease activation is
accompanied by dissociation of the accessory proteins. This thesis expands
upon current knowledge by focusing on structural comparisons of the (UreABC)3,
(UreABC-UreD)3, and (UreABC-UreDF)3 apoprotein complexes, and by
characterizing the UreG accessory protein.

The structure of urease apoprotein is known while those of the two
activation complexes are unknown. Computational analysis of the urease crystal
structure suggested that a hinge in the amino terminal region of UreB could allow
a repositioning of this subunit in one of these complexes to open up the nascent
active site. I tested the effects of hindering the flexibility of this peptide by
substituting two glycine residues with prolines, revealing an important role for

Gly11. In addition, the results of small angle X-ray scattering analyses allowed

me to conclude that the UreD and UreF accessory proteins are positioned at the
vertices of the urease structure, very close to the UreB subunit. Significantly, the
data were better fit to a model in which UreB was repositioned according to the
proposed conformational change, thus opening the active site for activation.

The UreG accessory protein, responsible for hydrolyzing GTP during the
activation process exists as an independent protein in addition to being part of
the activation complex. I created a biotin-tagged version of UreG, allowing for an
improved purification procedure. I showed that the biotin tag had no adverse
effect on UreG’s ability to activate urease and created a series of mutants of
UreG in which conserved residues were replaced by alanine. Most of the
mutations resulted in the complete loss of urease activity in vivo, although
substitution of Cys72, His74, and Ser111 (which correspond to metal ligands in
the related protein HypB) exhibited negligible changes in metal binding
parameters. In additional studies, I show the ability of the protein to form a novel
complex with urease apoprotein, UreD, UreF, UreG, and UreE. Furthermore, I
demonstrated that the D80A variant of UreG interacted only with UreE.
suggesting that Asp80 stabilizes the larger complex. The two new complexes

uncovered here set the stage for future studies involving their characterization.

A mis padres, por su apoyo incondicional.
A mi hermana Marcela y mi hermano Carlos, por su carifio.

A mi hija Mila, por sus besos, abrazos y sonrisas.

iv

ACKNOWLEDGEMENTS

This has been quite a journey. Life gave me the chance to become a better
scientist and a better person at the same time, which made it exhausting at times.
That's why, nothing presented here could have been possible without the
incredible people who surrounded me, starting with my family, to whom I
dedicated this dissertation. I was also very lucky to find a great mentor: Dr.
Robert Hausinger, Bob. He gave me the freedom, guidance and support I
needed to develop a project I believe in. Working with him was an immense
privilege that I will treasure forever. Bob was also very good putting together a
great group of people: Scott, our obligated first stop to learn about anything; Piotr
and Tina, two very knowledgeable people that were always willing to help with
suggestions and talk about life and food; Mukta, Kim, Jana, Meng, Thalia, Jodi,
Erick and Nicholas, my lab mates whom I shared so much time, ideas, jokes,
complaints, everything!; the best undergrads: Kimberly and Rachel, who helped
me with the experiments and not to get bored in the lab; Andrea, Bruce and
Aaron, whom I enjoyed getting to know.

I was also very lucky to belong to the biochemistry department in MSU,
were the graduate secretaries Jessica and Julie became my friends; my
committee members and other faculty who supported me: Dr. Christoph Benning,
Dr. Charlie Hoogstraten, Dr. Leslie Kuhn, Dr. Dennis Arvidson, Dr. Michael
Garavito, Dr. Michael Feig and Dr. Bill Wedemeyer; the program director, Dr. Jon
Kaguni and the former chair, Dr. Shelagh Ferguson-Miller. Also my classmates
James, Colleen, Cora, Chetan, Alec and Dean.

Finally my friends: Clarisa, Mariah, Andres, Karen and Soren, they made
my life easier and funnier, which was sometimes a big challenge.

To each of you, a piece of my heart.

Thank you.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................. ix
LIST OF FIGURES ............................................................................ x
ABBREVIATIONS .......................................................................................... xii
Chapter 1: Introduction .................................................................................... 1

INTRODUCTION TO NICKEL METABOLISM............................................2
1. General features of nickel incorporation into proteins........................2

1.1.Transport of Ni into the Cell ................................................... 2
1.2.Additional Processing of Ni in the Cell ...................................... 4
2. Nickel-containing enzymes and their activation process ...................... 6
2.1 . Hydrogenase .................................................................................. 6
2.2. Hydrogenase Activation ................................................................. 7
2.2.1. Metallochaperones: HypA, HypB, and SlyD ......................... 8
2.2.2. Hydrogenase Molecular Chaperones: Hpr and HypB ...... 10
2.3. Carbon Monoxide Dehydrogenase (CODH) ................................. 12
2.4. AcetyI-CoA Synthase/CODH (ACS/CODH) .................................. 14
2.5. CODH and ACS Activation ............................................................ 15
2.5.1. CODH Metallochaperone: CooJ .......................................... 15
2.5.2. CODH and ACS Molecular Chaperones: CooC/AcsF ......... 16
2.6. Methyl Coenzyme M Reductase ................................................... 16
2.7. Methyl Coenzyme M Reductase Activation ................................... 18
2.8. Superoxide Dismutase (SOD) ....................................................... 18
2.9. Glyoxylase ..................................................................................... 20
2.10. Aci-Reductone Dioxygenase (ARD) .......................................... 21
2.11. Other Potential Metallochaperones ........................................... 22
3. Urease ................................................................................................. 23
3.1 . Urease mechanism ....................................................................... 24
3.2. Urease activation .......................................................................... 25
3.2.1. Urease Metallochaperone: UreE ......................................... 26
3.2.2. Urease Molecular Chaperones: UreD, UreF and UreG ....... 31
CONCLUSIONS AND REMAINING QUESTIONS ........................................ 34
REFERENCES ............................................................................................... 35
Chapter 2: The Structure of Urease Activation Complexes Examined by Flexibility
Analysis, Mutagenesis, and Small-Angle X-Ray Scattering Approaches ....... 50
ABSTRACT .................................................................................................... 51
INTRODUCTION ............................................................................................ 52
EXPERIMENTAL PROCEDURES ................................................................. 55
Protein Purification .............................................................................. 55
Site-Directed Mutagenesis and Activity Assay...... 55
Metal Quantification .............................................................................. 56

vi

FIexibilityAnaIysis... 5.6

SAXS Measurements and Analysis ............................................. 57
Small- angle X- -ray scattering analysis and modeling ......................... 58
RESULTS ...................................................................................... 61
Flexibility Analysis of Urease61
Mutagenesis of Hinge Residues ................................................. 70
Small-Angle X-Ray Scattering Measurements and Analyses .......... 72
Models of the complexes ........................................................... 73
DISCUSSION .................................................................................. 80
ACKNOWLEDGMENTS .................................................................... 82
REFERENCES .................................................................................. 83
Chapter 3: Mutagenesis of the Klebsiella aerogenes UreG Urease Accessory
Protein: Effects on UreG Properties and Urease Activation ...................... 88
ABSTRACT ..................................................................................... 89
INTRODUCTION ................................................................................................ 90
EXPERIMENTAL PROCEDURES... ....94
Vector Construction Cell Growth, and PurIfcatIon Of BIotIn-Tagged
UreG... .................................................................................................... 94
Site-Directed Mutagenesis... 96
Circular Dichroism (CD)... 97
Analytical Gel Filtration Chromatography 98
Metal Quantification ................................................................... 98
Nickel Binding .......................................................................... 98
Analysis of Cells Expressing the Urease Operon Encoding the
UreGb Variants ........................................................................................ 98
Urease Activity Assay399
Pull-Down Assay599
Western Blot ........................................................................... 100
RESULTS ........................................................................................ 100
Characterization of Biotin-Tagged UreG (UreGb)......... 100
Targeting Residues for Mutagenesis... 103
Nickel Binding to UreG, UreGb and Variants” ..............104
Effect of UreGb Variants on Urease Activity In Cell ExtraCts .............. 106
Pull- down Assays ............................................................................. 108
DISCUSSION ................................................................................................ 112
ACKNOWLEDGMENTS .............................................................................. 114
REFERENCES .................................................................................. 115
Chapter 4: Additional studies, conclusions and remaining questions ......... 119
ADDITIONAL STUDIES ................................................................................ 120
1. Maltose binding protein-UreF (MBP-UreF) fusion protein crystallization
attempts .......................................................................................... 120
a. Expression and purification of MBP-UreF ..................... 120
b. Crystallization of MBP-UreF .......................................... 122

vii

2. Crystallizations attempts for UreG .................................................. 123

3. UreG homology model .................................................................... 124
CONCLUSIONS AND REMAINING QUESTIONS ........................................ 128
REFERENCES ............................................................................................... 132

Images in this dissertation are presented in color

viii

Table 2.1:
Table 2.2:
Table 2.3:
Table 2.4:
Table 2.5:
Table 2.6:
Table 3.1.
Table 3.2.
Table 3.3.

Table 4.1.

LIST OF TABLES

Polar Interactions of Region 1 (UreB residues 2 — 8)...............68
Hydrophobic Interactions of Region 1 (UreB residues 2 — 8) ..... 68
Polar Interactions of Region 2 (UreB residues 11 — 19)............68
Hydrophobic Interactions of Region 2 (UreB residues 11 — 19)..68
Polar Interactions of Region 3 (UreB residues 20 - 101) .......... 69

Hydrophobic Interactions of Region 3 (UreB residues 20—101)..69

Plasmids used in this study ................................................ 95
Oligonucleotides used to generate ureG mutations .................. 97
Thermodynamics of nickel ion binding to UreG proteins .......... 105
Conditions that showed UreG crystals or promising

precipitates ................................................................................................. 123

ix

LIST OF FIGURES
Figure 1.1. Generalized mechanism of metallochaperones and molecular
Chaperones .................................................................................... 5
Figure 1.2. Dinuclear Ni-Fe active site of a [NiFe] Hydrogenase ................. 7

Figure 1.3. Active sites of structuralIy-Characterized Ni-containing
enzymes ........................................................................................ 1 3

Figure 1.4. Crystal structure of K. aerogenes urease and Dinuclear Ni-Ni
active site of urease .......................................................................... 24

Figure 1.5. Proposed urease activation process ............................................ 26

Figure 1.6. K. aerogenes UreE (upper structure, PDB Code 1GMU) and B.
pasteurii UreE (PDB Code 1EAR) ........................................................ 30

Figure 2.1. Proposed pathway of urease activation...... 54

Figure 2.2. Tether and hinge regions between UreB and UreC from the
crystallographic structure of urease ................................................................ 64

Figure 2.3: Two views of (A) the native conformation of urease, (B) UreB
conformation 1 (torsionally adjusted UreB GIy11 and GIy18 residues), and
(C) UreB conformation 2 (severed linker, docked domain, and reconnected

linker) .............................................................................................. 66
Figure 2.4. Close-up of the repositioning of UreB from its crystallographic
position ............................................................................................ 67
Figure 2.5. Two pools of the UreB G11P mutant urease resolved by phenyl-
Sepharose chromatography ................................................................ 72
Figure 2.6. I(q) curves derived from the scattering data for urease ............... 74
Figure 2.7. P(r) curves derived from the scattering data for urease. ............. 75

Figure 2.8: Four views (two with UreABC in ribbon and two with UreABC in

spacefilling representation) of the best models of (UreABC-UreD)3 generated
by adding ellipsoids for UreD to the (A) native urease conformation, (B) UreB
conformation 1, and (C) UreB conformation 2 ......................................... 77

Figure 2.9. Predicted positioning of UreD and UreF relative to the
crystallographic structure of (UreABC)3, based on best-fit models to SAXS

data ............................................................................................. 80
Figure 3.1. Proposed urease activation process ....................................... 91

Figure 3.2. HypB dinuclear zinc site ..................................................... 93
Figure 3.3. UreGb purification ........................................................... 102
Figure 3.4. CD spectra for UreG and UreGb ........................................ 102

Figure 3.5. Size exclusion profile of native UreG and UreGb...................103
Figure 3.6. Metal binding to UreGb and selected variants.......................106
Figure 3.7. Analysis of mutant UreGb levels in cell extracts .................... 107

Figure 3.8. Urease activity in cell extracts expressing UreGb and its

mutants ........................................................................................ 107
Figure 3.9. Pull-down assays ........................................................... 110
Figure 3.10. Analysis of the UreE content in pull down samples ................ 111

Figure 4.1. SDS-PAGE of MBP-UreF expression on E. coli after induction.122

Figure 4.2. SDS-PAGE of purified MBP-UreF ............................................. 122
Figure 4.3. UreG model and HypB structure ................................................ 126

Figure 4.4. Partial superimposition of HypB crystal structure and UreG
Model ............................................................................................................ 127

xi

ACS

ARD

CD
CHa-S-COM
CoA-SH
COB-SH
CODH
Co-FeSP
COM-SH
CP

Glx

GSH

GTP

MBP

NMR

ORF

PAR

PDB
SAXS
SDS-PAGE
SOD
(UreABC)3

ABBREVIATIONS

acetyl-coenzyme A synthase

aci-reductone dioxygenase

circular dichroism

methyl-S—coenzyme M

coenzyme A

coenzyme B, N-7-mercaptoheptanoylthreonine phosphate
carbon monoxide dehydrogenase

corrinoid-iron-sulfur protein

2-thioethanesulfonate

carbamoyl phosphate

glyoxylase

glutathione

guanosine triphosphate

maltose binding protein

nuclear magnetic resonance

open reading frame

4-(2-pyridylazo)-resorcinoI

Protein Data Bank

small-angle x-ray scattering

sodium dodecyl sulfate polyacrylamide gel electrophoresis.
superoxide dismutase.

urease apoprotein

xii

(UreABC-UreD)3 complex of UreD bound to urease apoprotein
(UreABC-UreDF)3 complex of UreD and UreF bound to urease apoprotein

(UreABC-UreDFG)3 complex formed by UreD, UreF, and UreG bound to urease
apoprotein

xiii

CHAPTER 1

Introduction

Portions of this Introduction were derived from “Chaperones of Nickel
Metabolism”, Chapter 14 of Nickel and Its Surprising Impact in Nature (2007)
John Wiley & Sons, Ltd., written by
Soledad Quiroz, Jong K. Kim, Scott B. Mulrooney, and Robert P. Hausinger

INTRODUCTION TO NICKEL METABOLISM

Nickel is an essential micronutrient of many organisms where it serves as
a cofactor for enzymes involved in several critical metabolic processes (1, 2).
Like other transition metal ions, excess Ni is toxic to cells; thus, synthesis of
these Ni-enzymes requires the presence of carefully controlled Ni-processing
mechanisms that range from selective transport of Ni into the cells to productive
insertion of Ni into the apoproteins. Various accessory proteins participate in
these processes and are required for the biosynthesis of several Ni-dependent
enzymes. Here, I provide an overview of the catalytic activity, biological role, and
active site architecture of urease and briefly describe seven other structurally
Characterized Ni—dependent enzymes. In addition, I summarize what is known
about activation of these Ni-enzymes and emphasize two particular accessory
protein roles: metallochaperones that bind and deliver Ni to the apoprotein forms
of the enzymes, and molecular Chaperones that ensure productive conformations

of the apoproteins for Ni incorporation.

1. General features of nickel incorporation into proteins
1.1.Transport of Ni into the Cell
The first required step for synthesis of any Ni-enzyme is for the cell to take
up the metal ion from the environment in a regulated manner. Bacteria have
developed two major types of high-affinity Ni transport systems for efficient Ni

uptake (3, 4): ABC-type transporters and Ni-spec'rfic penneases.

The best-characterized ABC-type transporter system is that encoded by
the E. coli nikABCDE operon (5), which is regulated by the product of a
downstream gene, nikR. NikA is a periplasmic Ni-binding receptor protein with 3
Ni dissociation constant (Kd) of less than 0.1 pM (ten-fold lower than for Co, Cu,
or Fe) (6). Two crystal structures of metal-bound NikA reveal pronounced
differences at the metal-binding site: in one case, a penta-hydrated Ni ion is
suggested to be bound (with a single polar interaction) within a large cavity of the
protein (7), whereas the second study reported the binding of a monohydrated
Fe-EDTA complex at the same site of the protein using many specific
interactions between the chelator and the protein side chains (8). The long (2.7
A) average Ni-O bond distance of the first crystal structure is inconsistent with
spectroscopic data suggesting a much shorter distance (2.06 A) (9), whereas the
latter structural data more easily accommodate this distance. It is likely that a yet
unidentified chelated form of Ni, rather than the ion itself, is the physiological
species recognized by NikA. NikB and NikC are hydrophobic transmembrane
proteins forming a pore for passage of the metal. NikD and NikE bind and
hydrolyze ATP, and couple this energy release to the transport process. Ni
homeostasis is achieved by use of NikR, a Ni-specific transcriptional repressor
that binds to the NikR box in the promoter of the nik operon in the presence of Ni,
resulting in suppression of Ni uptake (10).

The first Ni-specific permease (encoded by hoxN) was identified in
Ralstonia eutropha (11). HoxN is an integral membrane protein containing eight

membrane-Spanning segments according to membrane topology analyses, and

is the prototype of a novel family of transition metal perrneases. Transport assays
showed that HoxN has a high affinity for Ni with a transport constant (Kt) of ~ 20
nM, but with very low capacity. HoxN homologues have been reported in many
bacteria (e.g., HupN in Bradyrhizobium japonicum, NixA in H. pylon’, NicT in
Mycobacten’um tuberculosis, and Nth in Rhodococcus rhodochrous) and Nic1 p
in the fission yeast, Schizosaccharomyces pombe (12). The absence of HupN
resulted in low levels of hydrogenase activity in B. japonicum under Ni-Iimiting
conditions. Nic1p and NixA are essential for urease activity, and NixA exhibits a
Ni K, of 11 nM. Nth was originally identified as a Co transporter in R.
rhodochrous J1, but subsequent reinvestigation revealed that the permease
transports both Ni and Co with a slight preference for Co. Regulation of the

genes encoding these perrneases is not well studied.

1.2.Additional Processing of Ni in the Cell

Once Ni enters the cell, it must be delivered and incorporated into the
correct binding sites of Ni enzymes. This process may require
metallochaperones, molecular Chaperones, and a variety of other assembly
steps.

The term metallochaperone refers to a protein that reversibly binds a
metal ion, transports it within the cell, and provides it for metallocenter assembly
to the target apoprotein. Molecular Chaperones are proteins that prevent
misfolding or assist in re-folding of other proteins, often by using energy derived

from nucleotide hydrolysis. For example, the best studied molecular Chaperones

are the GroES:GroEL chaperonin, Hsp70, and Hsp40 that act on many cellular
proteins (13, 14). Recent studies have shown that SlyD, a Ni-binding protein,
similarly exhibits a diverse molecular Chaperone role (15-18). Such non-specific
molecular Chaperones are likely to stimulate the proper folding of many Ni-
containing enzymes, as evidenced by the diminished hydrogenase activity in
ngL or ngS mutants and by the specific binding of GroEL to the Hch
precursor protein (19). While these housekeeping proteins are rather non-specific
in their action, this section focuses on molecular Chaperones that are specific to
individual Ni-enzyme activation systems. These proteins may drive the reaction
by coupling metal insertion to nucleotide hydrolysis and/or they may use a
metallochaperone rather than the free metal ion. In general, such proteins appear
to be more essential to the activation processes than the metallochaperones,
whose absence often can be overcome by excess Ni. A general scheme for

metallochaperone and molecular Chaperones function is presented in Figure 1.1.

 

Holoprotei
I Molecular Chaperonj l Metallochaperona

Ni I
Target 3"
Apoprotein (NTP)
I Ni-Metallochaperona
I ApoproteinzMolecuIar Chaperoni

Figure 1.1. Generalized mechanism of metallochaperones and molecular
Chaperones. NTP, nucleotide triphosphate; required in some, but not all,
molecular Chaperones.

 

 

 

 

 

 

 

Accessory proteins can be involved in several other processes. For
example, apoprotein proteolysis is associated with synthesis of hydrogenases
and Ni-SOD. Cofactor synthesis is required prior to incorporation of the F430
tetrapyrrole into methyl coenzyme M reductase. Finally, many enzymes require
the incorporation of another constituent prior to addition of Ni, such as the lysine
carbamate of urease, the Fe(CN)2(CO) site of hydrogenase, and the iron-sulfur
cluster components of CODH and ACS. In the following sections, I discuss the

specifics of these processes for urease and then for selected other examples.

2. Nickel-containing enzymes and their activation process
2.1 . Hydrogenase
Hydrogenases catalyze the reversible oxidation of molecular hydrogen into
protons and electrons (Eq. 1.1). These enzymes provide a mechanism for many
microorganisms to use H2 as an energy source by generating a proton gradient
or to remove excess reducing power in the form of molecular hydrogen (20).
H2 2 2H++2e' (1.1)

Three distinct Classes of hydrogenases are defined by the metal content of
their active sites: [NiFe]-hydrogenases, [Fe]-hydrogenases, and [iron-sulfur-
cIuster-freej-hydrogenases (20, 21). The crystal structures of several [NiFe]-
hydrogenases have been resolved, including those of Desulfovibn‘o gigas and
Desulfomicrobium baculatum (22-24). Each heterodimeric protein has three iron-
sulfur clusters in its small subunit and a [NiFe] active site in its large subunit. The

active center contains Ni coordinated by four Cys residues (or three Cys and a

selenocysteine in the D. baculatum enzyme), two of which bridge to the Fe that is

also liganded by one carbon monoxide and two cyanide groups (Figure 1.2).

 

Figure 1.2. Dinuclear Ni-Fe active site of the [NiFe] hydrogenase from
Desulfovibn’o baculatus (PDB code 1001). The Ni is bound to a seleno-Cys and
three Cys (or to four Cys in related enzymes), two of which also coordinate the
Fe. The Fe—bound diatomic ligands are two cyanide and one carbon monoxide
molecules.
2.2. Hydrogenase Activation

Seven accessory proteins are required to synthesize the Escherichia coli
HyCGE NiFe-hydrogenase (25), the paradigm system for defining the activation
process of these enzymes (26, 27). These accessory proteins are the products of
the six hyp genes (hypABCDEF) and another gene encoding a specific
endopeptidase (hycl). The current model of Hch (large subunit) maturation
includes a complicated series of steps involving (1) HprEF-mediated formation
of an Fe(CN)2(CO) site in a process facilitated by Hpr (28); (2) insertion of Fe

and its ligands into the precursor of the large subunit (retaining its C-ten'ninal

extension) when in complex with Hpr (29); (3) GTP-dependent addition of Ni to

the active center mediated by HypAB; and (4) proteolytic processing of the C-

terrninus of Hch by Hbe, leading to internalization of the catalytic center.

2.2.1. Metallochaperones: HypA, HypB, and SlyD

Of the many proteins involved in maturation of [NiFe] hydrogenases, three
are known to directly bind Ni and may function as metallochaperones: HypA,
HypB, and SlyD.

HypA designates the ~13-kDa protein required for activation of
hydrogenase 3 of E. coli and the corresponding protein in many other
hydrogenase systems. Homologues are termed HupA or Hbe when referring to
the protein used for E. coli hydrogenase systems 1 and 2. Purified HypA from H.
pylori binds two Ni ions in a cooperative manner (30). Site-directed mutagenesis
studies revealed a single His residue is required for binding Ni, and introduction
of the corresponding His to Ala mutation resulted in substantial loss of
hydrogenase activity (30). Subsequent investigations of E. coli HypA showed that
it binds stoichiometric amounts of Ni and Zn, with pM and nM affinities,
respectively (31). Based on UV/visible spectroscopic results indicating thiolate
ligation, the bound Zn is proposed to have a structural role. Similar mutagenesis
and binding studies of E. coli Hbe found a single histidine is necessary for Ni
binding, but mutagenesis of this residue resulted in a protein that retained the
ability to bind Zn (32).

HypB, alternatively termed HupB in certain microorganisms, is a ~30—kDa

protein that contains a nucleotide-binding motif and possesses low levels of

GTPase activity (30, 33-36). Site-directed mutations in the GTP-binding motifs
result in elimination of hydrogenase activity (34). In addition to its GTPase
activity, HypB proteins of selected organisms contain His-rich regions that are
capable of binding several Ni ions. When this motif is deleted from B. japonicum
HypB, the protein still binds one equivalent of Ni and retains competence in
activating hydrogenase (35, 37, 38). The best-studied HypB is that from E. coli.
This protein lacks a His-rich region, yet it tightly binds one Ni per monomer (Kd of
0.12 pM) using a CXXCGC motif at the N-tenninus (39). Furthermore, the E. coli
protein has a second metal-binding site located in the GTP-binding domain that
has weaker affinity for either Ni or Zn ions (39).

In addition to the myriad studies of the individual HypA and HypB proteins
and their homologues, there is significant evidence that the two proteins interact.
Chemical cross-linking studies showed that a stable HypA-HypB complex is
formed for these proteins (30) (31). In contrast to these results, chemical cross-
Iinking studies carried out with a Strep-tagged variant of Hbe failed to detect an
association with HypB (32). Although the above studies of HypA and HypB
proteins have greatly added to our knowledge, the question of how they function
in Ni delivery and/or insertion into hydrogenase remains to be discovered.

SlyD is a ~21-kDa protein possessing an N-terrninal region (146 amino
acids with similarity to FK506-binding proteins) that contains peptidyl-prolyl
cis/trans-isomerase activity and a Short C-terminal metal-binding region rich in
His, Asp, Glu, and Cys (40, 41). Evidence suggests that SlyD may act as a

Chaperone for several proteins (15-18). More pertinent to this discussion, SlyD

reversibly binds Ni in its C-terminal region such that the peptidyllprolyl isomerase
activity is inhibited (41). The ability of SlyD to bind Ni was suggested by its co-
purification with several recombinant His-tagged proteins when using Ni-
nitrilotriacetic acid affinity chromatography (17, 42-44). Of particular interest with
regard to maturation of the [NiFe] hydrogenases, E. coli SlyD was shown to
interact with HypB from the same source (45). Furthermore, cells deleted in stD
have greatly reduced activity of all three hydrogenases and reduced intracellular
concentrations of Ni. These findings suggest that SlyD has a role in the Ni

insertion step of hydrogenase activation (45).

2.2.2. Hydrogenase Molecular Chaperones: Hpr and HypB

The complex biosynthetic pathway of E. coli hydrogenase 3 includes two
molecular Chaperone-like proteins: Hpr and HypB. E. coli contains a second
Hpr-like protein, termed HybG, which specifically binds to hydrogenase 2 (both
Hpr and HybG bind to hydrogenase 1, but only the former facilitates activation)
(46). Homologues to Hpr and HypB are found in many other organisms where
they are sometimes given alternative designations (e.g. HupB and HupC). Hpr
(or its homolog) plays a central and multifaceted role in [NiFe]-hydrogenase
biosynthesis. Hpr forms a complex with Hpr, especially when the synthesis of
CP is reduced so that the formation of the Fe(CN)2(CO) center is hindered (29).
The Hpr-Hpr species additionally binds HypE in such a manner as to allow its
carbamoylation by Hpr (28). Hpr dissociates from Hpr as a new complex,

Hpr-Hch, is formed (47, 48) containing the Fe(CN)2(CO) center, formed in the

10

earlier complex, but still lacking Ni (47). The interaction between the Chaperone
and the large subunit precursor requires the N-terminal Cys residue of Hpr and
a particular large subunit Cys residue, which eventually coordinates Ni in the
active site (49). Ni is provided by the action of HypA/HypB metallochaperone
(50), with HypB additionally having a molecular Chaperone role. After the
complete set of metallocenter components is in place, Hpr dissociates, Hycl
binds to the Hch-bound Ni and becomes proteolytically active (51). The large
subunit extension is removed, resulting in the [NiFe] site becoming buried in the
protein.

Nickel insertion into the Fe(CN)2(CO)-containing and Hpr-bound
hydrogenase precursor requires the HypA/HypB metallochaperone, with HypB
also exhibiting a GTP-dependent molecular Chaperone role (34). HypB is
homologous to the urease accessory protein UreG and likewise contains a
nucleotide-binding motif. Native HypB exhibits weak GTPase activity, but a
mutation affecting the P-loop eliminates the GTPase activity and greatly
decreases the hydrogenase activity (30, 52). The HypB proteins of some
organisms contain His-rich termini that are able to bind Ni; removal of this
sequence has only a small effect on hydrogenase activity while having a larger
affect on cellular Ni content. The HypB molecular Chaperone appears to drive Ni
insertion into the hydrogenase subunit by coupling this reaction to GTP

hydrolysis (30, 33-36).

11

2.3. Carbon Monoxide Dehydrogenase (CODH)

CODHS catalyze the reversible oxidation of carbon monoxide to carbon
dioxide (Eq. 1.2). Organisms possessing these enzymes play critical roles in
the global carbon cycle and the degradation of environmental pollutants (53).

CO + H20 2 002 + 2H+ + 2e‘ (1.2)

Crystal structures are known for CODHS from Carboxydothennus
hydrogenoformans and Rhodospin'llum rume (54, 55). Both proteins are ~ 130-
kDa homodimers containing five metal-sulfur clusters of three types (B, C, and D)
in a C-B’-D-B-C’ arrangement where the D cluster bridges the two subunits.
While the B, B’ and D sites are the same cubane type [4Fe-4S] clusters in both
proteins, the structures of the active site clusters (C and 0’) Slightly differ in the
two proteins. The C cluster of R. rubrum is essentially a [1Ni-3Fe-4S] cubane
bridged to a mononuclear Fe site, whereas the structure of the C.
hydrogenoformans enzyme can be viewed as a [3Fe-4S] cluster fused with a [Ni-

S-Fe] fragment containing a bridging sulfide (Figure 1.3A).

12

 

Figure 1.3. Active sites of structurally-characterized Ni-containing enzymes. In
each case, Ni is a solid black sphere, nitrogen atoms are blue, sulfur orange,
oxygen red, and carbon white. A. [Ni-Fe4-Ss] cluster of Carboxydothermus
hydrogenoformans CODH (PDB code 1SU8). The structure of this cluster slightly
varies in other CODH sites. B. [4Fe-4$]-Ni-Ni site of Carboxydothennus
hydrogenoformans ACS (PDB code 1RU3). The fourth ligand on the central Ni is
water. C. F430 Ni-tetrapyrrole of Methanobacterium thermoautotrophicum methyl
coenzyme M reductase (PDB code 1MRO). D. The active site of Streptomyces
coelicolor Ni-SOD (PDB code 1T6U). The imidazole nitrogen of His1 is a ligand
in the active enzyme, when the Ni is oxidized. E. E. coli Ni-glyoxylase showing
two bound water molecules (PDB code 1F9Z). HisS and Glu56 are derived from
one subunit and His74 and Glu122 from the second subunit in the symmetric
dimer. The two water molecules are displaced by substrate. F. Ni—COmaining form
of ARD from Klebsiella oxytoca as derived by a combination of solution structure
analysis and homology modeling (PDB code 1M40). The non-side chain ligands
of the metal are water molecules.

2.4.AcetyI-CoA Synthase/CODH (ACS/CODH)
The CODH activity described above is found in another set of enzymes isolated
from acetogenic bacteria and methanogenic archaea. The ACS/CODHs are
bifunctional catalysts that exhibit the activity shown in Eq. 1.2 and additionally
synthesize (or decompose) acetyI-coenzyme A (CoA-SH) using the remarkable
chemistry shown in Eq. 1.3. The CODH site of ACS/CODH reduces CO; to CO
and then this gaseous molecule traverses a molecular tunnel within the protein to
reach the ACS site where it is joined to CoA-SH and the methyl group from the
corrinoid-iron-sulfur protein (Co-FeSP). Along with the monofunctional CODHS,
these enzymes play a major role in the global carbon cycle and in the formation
and removal of greenhouse gases (56).
CD + CoA-SH + CH3-Co(lll)-FeSP Z CH3C(O)-S-COA + Co(l)-FeSP (1.3)
Crystallographic studies of Moore/Ia thermoacetica ACS/CODH revealed
that the tetrameric protein contains the dimeric CODH subunits at its core and
one ACS subunit on each end (57, 58). The ACS metallocenter is a [4Fe-4S]-Ni-
Ni site called the A-cluster. The [4Fe-4S] cluster is bridged to one Ni via a Cys
side chain, and this metal is in turn bridged by two Cys residues to a second Ni,
that is additionally bound by two backbone amides. The central Ni in the A-
cluster is subject to metal substitution, resulting in inactive Cu-Ni and Zn-Ni
species that were critical to identifying closed and open conformations of the
protein. The [4Fe-4S]-Ni-Ni cluster (Figure 133) also was observed in the

structure of the monomeric C. hydrogenoformans A08 (59).

14

2.5. CODH and ACS Activation

Information regarding the mechanism of Ni insertion into CODH is
available for R. rubrum where the cooCTJ gene cluster (60), located downstream
of the 0008 structural gene, is known to be involved. The CooC protein, which
contains a nucleotide-binding motif, acts as an ATP/GTP-dependent molecular
Chaperone, while CooJ delivers Ni by using its histidine-rich C-terminal motif.

Little is known about the mechanism concerning metallocenter assembly
in ACS/CODHS. Since the enzyme has two different sets of Ni-Containing active
sites, it is anticipated that several accessory proteins are required for
biosynthesis. Consistent with this notion, ACS/CODH gene clusters contain
several non-subunit open reading frames (ORFS). In particular, AcsF encodes a

CooC-like protein that is further described below.

2.5.1. CODH Metallochaperone: CooJ

The R. rubrum protein CooJ contains 115 residues of which 16 of the C-
terrninal 34 amino acids are His (61), an arrangement similar to the His-rich
regions in sequences of some HypB and UreE proteins. Cells containing a
chromosomal deletion that eliminates the His-rich region display Ni-dependence
for growth on CD that is identical to wild-type strain, while cells with an insertional
mutation of cooJ required 1000-fold higher Ni than wild type for optimal growth
(60). These findings suggest that the His-rich C-terminal region is not required for

CooJ function, and the functional Ni-binding site lies elsewhere in the protein.

15

2.5.2. CODH and ACS Molecular Chaperones: CooC/AcsF

The CODH operon of R. rubrum encodes the suspected molecular
Chaperone 0000. This membrane-bound homodimer of 62 kDa is related in
sequence to UreG of urease activation and HypB of hydrogenase biosynthesis
(60). CooC contains a P-loop motif in its N-terrninus, and the purified protein
hydrolyzes both GTP and ATP with similar Km values, but with a 10-fold greater
V"...x for ATP. Mutation of residues in the P-Ioop motif prevents Ni insertion into
CODH and abolishes the ATPase activity, both in vivo and in vitro. Ni is not
present in the purified protein (62).

The gene cluster encoding the ACS/CODH bifunctional enzyme of M.
thennoacetica includes a gene encoding AscF that resembles 0000 and the
other nucleotide-dependent molecular Chaperones described above. This protein
contains a P-loop and has five conserved Cys in a motif characteristic of iron
coordination. Despite the suspected importance of this gene for ACS/CODH
activation, its deletion had no effect on enzyme activity. It remains possible that a

second copy of the gene is present in the genome (63).

2.6. Methyl Coenzyme M Reductase
Methyl coenzyme M reductase catalyzes the reaction of methyl-S-
coenzyme M (CH3-S-CoM, where COM-SH is 2-thioethanesulfonate) with
coenzyme B (COB-SH, N-7-mercaptoheptanoylthreonine phosphate) to form

methane and the heterodisulfide, CoM-S-S—COB (Eq. 1.4). This is the final step of

16

methane formation in methanogenic archaea growing on simple molecules such
as acetate, methanol, formate, and carbon dioxide plus hydrogen gas (64).

CHa-S-COM + COB-SH —> CH4 + CoB-S—S-COM (1.4)

The X-ray crystal structure of methyl coenzyme M reductase, first obtained
from Methanothennobacter marburgensis (65), reveal that the protein is a 300-
kDa heterohexamer of three different subunits (dzfizvz) containing two molecules
of the Ni-containing tetrapyrrole, F430 (Figure 1.3C). This cofactor, named on the
basis of its Characteristic absorbance maximum at 430 nm when in the Ni(ll)
state, must be in the Ni(l) state for the enzyme to be active. Each active site F430
is buried deep in the protein and accessible from the surface by a 50 A long
channel composed of mainly hydrophobic amino acids through which CH3-S-
CoM can enter, and which is blocked by the binding of COB-SH. An interesting
aspect of this enzyme is the presence of five post-translationally modified amino
acids near the active site: thio-Gly, N-methyl-His, S-methyl-Cys, 5-methyl-Arg,
and 2-methyI-Gln. Labeling studies have shown that the methyl groups are
derived by methyl group transfer from S-adenosylmethionine, and not from the
methyl group of CH3-S-COM.

An enzyme of related interest is found in methanotrophic archaea (66),
such as those located in microbial mats that catalyze the anaerobic oxidation of
methane (67). These prokaryotes, closely related to methanogens in the order
Methanosarcinales, contain homologues of genes encoding methyl coenzyme M

reductase (68) and possess an F430-Iike molecule with a 46 Da mass increase

17

(67). The mechanism by which these microbes essentially reverse the last step

of methanogenesis remains unclear.

2.7. Methyl Coenzyme M Reductase Activation

The biosynthetic pathway of F430 is an offshoot of those for other biological
tetrapyrroles (69). Early labeling studies demonstrated that F430 is derived from
dihydrosirohydrochlorin, which is also the precursor of siroheme and corrinoids.
The dihydrosirohydrochlorin is formed from 5-aminolevulinic acid via
uroporphyrinogen Ill. The conversion of dihydrosirohydrochlorin to F430 requires
several steps including amidation of acetate groups on two rings, reduction of
two double bonds, cyclization of an acetamide to form the five-membered ring,
cyclization of a propionic acid to form the six-membered ring, and insertion of Ni.
However, the order of these steps and the mechanism underlying the Ni insertion

and F430 incorporation into the protein remain unknown.

2.8. Superoxide Dismutase (SOD)

SODs are ubiquitous metalloenzymes that function to protect biological
molecules from oxidative damage by catalyzing the dismutation of superoxide
anion radicals to peroxide and molecular oxygen (Eq. 1.5). In addition to the well-
known Cu,Zn-, Fe-, and Mn-containing 8003, recent studies have revealed the
existence of Ni-SODS in Streptomyces species and some cyanobacteria.

2 02- + 2 H+ —> H202 + 02 (1.5)

18

Crystal structures of Ni-SODS have been solved for S. seoulensis and S.
coelicolor enzymes (70, 71). The proteins are homohexamers consisting of four-
helix bundle subunits. The N-terminal loop coordinates the active site Ni(lll) in
square pyramidal geometry using two thiolate side chains (Cys-2 and Cys-6), two
backbone amides (His-1 and Cys-2), and the His-1 side chain ligand at the axial
position. The axial ligand is lost in the reduced state, with Ni(l|) becoming square
planar (Figure 1.30). Apoprotein structures show that the residues involved in
binding Ni are disordered.

Ni-SODs in Streptomyces species are products of sodN, which encodes a
preprotein with an N-terminal extension of 14 amino acids. During SOD
maturation, proteolytic cleavage precedes Ni binding and results in the creation
of the six-residue Ni-binding site. Recently, ORFS with significant similarity to NI-
8003 were identified in the genomes of several cyanobacteria including
Prochlorococcus man'nus MIT9313 (72). In this microbe, an ORF located
downstream of sodN and named sodX was suggested to be the peptidase for
maturation of the Ni-SOD. Coexpression of sodX and sodN in an oxygen-
sensitive E. coli strain restored oxygen tolerance in a Ni-dependent manner,
indicating the production of a catalytically active enzyme and providing
confirmatory evidence for the importance of SodX in Ni-SOD maturation. Ni-SOD
activity in S. seoulensis is stimulated by the overproduction of Cbith, a Ni-
binding protein, suggesting that it too many function in metallocenter assembly
(73). Contrary to this notion, the gene encoding Cbith is located between two

genes suggested to function in cobalamin biosynthesis. Further studies are

19

needed to elucidate the detailed maturation steps of Ni-SOD activation, including

the mechanism of Ni incorporation to the enzyme.

2.9. Glyoxylase

Glyoxylase I is the first of two enzymes in the pathway to convert cytotoxic
methylglyoxal into non-toxic q-hydroxycarboxylic acids. It converts the
hemimercaptal substrate, formed nonenzymatically from methylglyoxal and
glutathione (GSH, Eq. 1.6), to non-toxic S—D-lactoylglutathione (Eq. 1.7), which is
the substrate for Glyoxylase lI (Eq. 1.8). These enzymes are important for
cellular protection because methylglyoxal can exert toxic effects by reacting with
DNA, RNA and proteins.

CH3-CO-CHO + GSH Z CHg-CO-C(OH)-SG (1.6)

CH3-CO-C(OH)-SG —+ CH3-CH(OH)-CO-SG (1.7)

CH3-CH(OH)-CO-SG + H20 —> CH3-CH(OH)-COOH + GSH (1.8)

Unlike the case for glyoxylase l of humans, Saccharomyces cerevisiae,
and Pseudomonas putida, where the active site metal is zinc, glyoxylase I from
E. coli is completely inactive in the presence of Zn and is maximally active with Ni
(74). Reduced activity is found in the enzyme substituted with Co, Cd, and Mn.
Crystallographic analyses revealed that catalytically active forms of E. coli
glyoxylase l with Ni, Co, and Cd each have octahedral metal coordination (Figure
1.3E), which is also observed in the Zn-containing human enzyme, whereas the
inactive Zn-containing E. coli protein displays a five-coordinate metal site (75).

Several other pathogenic microorganisms are hypothesized to possess a Ni-

20

containing glyoxylase on the basis of sequence comparisons (76). The cellular

mechanism of Ni incorporation into glyoxylase l is unknown.

2.10. Aci-Reductone Dioxygenase (ARD)

Many microorganisms utilize the methionine salvage pathway to regenerate
methionine from methylthioadenosine, produced during polyamine biosynthesis
from S—adenosylmethionine. Aci-reductone is a key intermediate of this pathway,
and is oxidized to two different sets of products in Klebsiella pneumoniae. One
oxidation pathway leads to production of forrnate and the ketoacid precursor of
methionine. The other route of oxidation, a non-productive pathway, converts the
aci-reductone to formate, carbon monoxide, and methylthiobutyric acid.
Remarkably, the two reactions are carried out by the same enzyme, ARD,
depending on which metal is bound at the active site (Fe or Ni, respectively) (77).

The solution structure of K. pneumoniae Ni-ARD was determined by NMR
methods (78). The enzyme is a monomer containing two B-sheets that hinge
together to form a jellyroll. Unfortunately, paramagnetism of the bound Ni causes
broadening of the 1H resonance lines from residues near the metal center, thus
hindering the structural characterization of the active site. Biophysical studies
suggest the presence of three His ligands to the Ni, along with three other
nitrogen or oxygen atoms (79). Homology modeling of the active center, based
on the structure of jack bean canavalin (another member of the cupin family),
provides a reasonable model of the active site (Figure 1.3F). The mechanism of

Ni insertion into the enzyme is unknown.

21

2.11. Other Potential Metallochaperones

Several other proteins possess His-rich regions and/or tightly bind Ni;
however, none of these has been convincingly shown to facilitate Ni
metallocenter assembly. For example, Cbith of S. seoulensis contains a
carboxyl terminus in which 11 of 19 residues are His, and the cellular
overproduction of this protein stimulated Ni-SOD activity (73). On the other hand,
this protein is more likely to be involved in cobalamin biosynthesis on the basis of
flanking genes and its presence in cells that lack a Ni-SOD. The Hpn protein of
H. pylori is worth a few comments because of the high levels of Ni-containing
urease and the important role of hydrogenase in this microorganism (80, 81).
Hpn is comprised of only 60 amino acids, 28 of which are His (82). Deletion of
the corresponding gene has no effect on urease activity for cells grown on blood
agar, but does make the cells more susceptible to growth inhibition at high Ni
concentrations (83). Recent studies have shown Hpn binds 5 Ni per monomer
(K, 7.1 pM) and provided evidence that the concentrations of this protein
correlate to the intracellular Ni concentration and to the cell’s ability to tolerate
high Ni concentrations (84). The authors suggested that Hpn may function in Ni
storage, Ni donation, and Ni detoxification. It will be of interest to learn whether
follow-up studies confirm the putative metallochaperone role for this protein and
to monitor whether Ni metallochaperones for other enzyme systems are

idenfified.

22

3. Urease

Urease catalyzes the hydrolysis of urea to produce ammonia and
carbamate. The latter molecule spontaneously decomposes to yield another
molecule of ammonia and carbonic acid (Eqs. 1.9 and 1.10). This enzyme, found
in plants, fungi and bacteria, has several biological roles including its participation
in recycling of environmental nitrogen and its use as a virulence factor in
pathogenic microorganisms that are associated with gastric ulceration and
urinary stone formation (85).

H2N-CO-NH2 + H2O -—+ H2N-COOH + NH3 (1.9)

H2N-COOH + H2O —+ H2CO3 + NH3 (1.10)

Crystallographic analyses have revealed that most bacterial ureases
possess three structural subunits (encoded by ureA, ureB, and ureC) associated
into a trimer of trimers [(UreABC)3] (Figure 1.4, left), with each UreC subunit
containing a dinuclear Ni active site bridged by a carbamylated Lys residue (86-
88) (Figure 1.4, right). Some species, such as Helicobacter pylori, have only two
subunits (UreA, corresponding to a fusion of the small subunits in other bacteria,
and the large subunit, labeled UreB) in a (UreA3UreBg)4 supramolecular structure
(89). Plants and fungi have a single subunit corresponding to a fusion of all of the
bacterial subunits, and form an Urea urease structure (90) that resembles a dimer

of the bacterial enzyme.

23

 
 
 

   

L331

  
 

    

   

   
 

., —v .3 ...
‘ , a I .
CA I “ ‘.
«3’ fish;
. " Lys217*
- "‘ His246
His134
I ’7
~ -0:
~ His136
Hi5272
Asp360

Figure 1.4. Left: Crystal structure of K. aerogenes urease. UreA is colored in
yellow, UreB in red and UreC in blue. The green spheres are the nickel ions at
the active site. Right: Dinuclear Ni-Ni active site of urease (PDB code 1FWJ).
The metal-bridging side chain is a carbamylated Lys and the three red spheres
coordinated to the metals are water molecules.

3.1. Urease mechanism

The breakdown of urea requires access of the molecule into the buried active
site. It has been suggested that a conformational change in the flap formed by
residues 308-336 of UreC could allow easy access of the substrate to the active
site (86, 91). Two mechanisms have been proposed for urease, with both
initiating by urea displacement of a water molecule coordinated to NH (the one
bound by His246 and His272). Then in one case, a water molecule bound to Ni2
attacks urea to form a tetrahedral intermediate and His320 protonates the amido
nitrogen, promoting the formation of ammonia and carbamate, the final products

of the reaction. The alternative mechanism suggests that the oxygen from urea

coordinates to NH and one amido group to Ni2; then, the bridging water attacks

24

the carbonyl group to form a tetrahedral intermediate and donates the proton to
the distal amido group, with Asp360 assisting the protonation (92). Until now,
there is no evidence to Clarify the origin of the water molecule attacking urea and
distinguishing whether the amido group binds to Ni2.

3.2. Urease activation

The urease gene cluster of most bacteria is composed of both structural
genes (ureABC) and accessory genes (typically including ureDEFG, with
additional urease-related genes present in some species). The structural gene
products assemble into an apoprotein that requires activation by the accessory
proteins. The best—studied urease activation system is that found in Klebsiella
aerogenes, which contains the ureDABCEFG gene cluster (93, 94). Using this
system, UreD, UreF, and UreG were identified as forming a GTP-dependent
molecular Chaperone that binds urease apoprotein (95), while UreE was shown
to function as a metallochaperone that delivers Ni (96, 97). A scheme that shows

this process is depicted in figure 1.5.

25

Active
Urease

    

0
+ 3GDP + 3Pi
Figure 1.5. Proposed urease activation process. The K. aerogenes UreA, UreB
and UreC urease subunits assemble into the (UreABC)3 apoprotein (depicted
simply as a trimeric species since UreA plus UreB or all three subunits are fused
together in ureases from some sources). UreD, UreF and UreG sequentially bind
to form the (UreABC-Ure0)3, (UreABC-UreDF)a. and (UreABC-UreDFG)3
activation complexes. 002 adds to the active site Lys as Ni” ions are delivered
to (UreABC-UreDFG)3 by the dimeric UreE metallochaperone in a process that
requires GTP hydrolysis, with UreE and (UreDFG)3 being released from the
activated urease.

3.2.1. Urease Metallochaperone: UreE

Among the multiple accessory genes required for urease activation in
most urealytic organisms, UreE appears to function as a metallochaperone that
delivers Ni to the urease apoprotein. The first suggestion that UreE functions as
a Ni-binding protein was provided by the sequences of the K. aerogenes and
Proteus mirabilis urease operons (93, 98). The carboxyl termini of these proteins
contain His-rich regions consisting of 10 His in the last 15 residues for K.
aerogenes and 9 of the last 10 residues in the case of P. mirabilis, indicative of a

potential metal binding role. Subsequent sequences of ureE genes from other

26

sources reveal that the His-rich C-terminal region is common, but it is completely
absent in some organisms (99). Equilibrium dialysis studies of K. aerogenes
UreE showed that about 6 Ni bind per dimeric protein (100), while metal-binding
studies of Bacillus pasteun'i UreE, which contains only two conserved His
residues in this region, found a single Ni bound per dimer (101). Purified UreE
proteins also bind other metal ions, such as Cu and Zn, demonstrating that the
specificity of urease for Ni does not reside solely with this delivery protein (102).
Using site-directed mutation methods to create a truncated form of K. aerogenes
UreE with the last 15 amino acids removed (His144*UreE), the His-rich region
was demonstrated to be non-essential; i.e., the truncated protein still binds 2-3 Ni
ions per dimer and is still competent in facilitating Ni-dependent activation of
urease in vivo (103, 104). In a complementary study, the native H. pylori ureE
gene, which does not encode a protein with a His-rich C-terminal tail, was fused
to several extensions to produce different His-rich regions (105). The resulting
His-rich variants show increased Ni-binding and cells containing these variants
have increased urease levels; thus, the C-terminal His-rich region has a Ni-
sequestering function that aids in urease activation.

Several lines of evidence indicate that UreE interacts with other accessory
proteins during Ni-dependent activation of urease. In vitro studies showed that a
complex of urease apoprotein with UreD, UreF, and UreG is fully activated only
by including UreE in a mixture containing GTP, bicarbonate, and Ni (97), thus
providing strong evidence that UreE functions as a metallochaperone to deliver

Ni to the UreDFG-urease apoprotein complex. These studies also showed that

27

UreE does not simply function as a reversible Ni-binding protein because
activation occurred even when metal ion chelators were included in the reaction
(97). Additional work involving yeast two-hybrid analysis demonstrated an
interaction between H. pylori UreE and UreG proteins (106, 107).

Site-directed mutagenesis and structural studies provided detailed insights
into the metal-binding properties of UreE. Variants of K. aerogenes His144*UreE
affecting His-110 or His-112 exhibit reduced Ni binding while not greatly affecting
urease activation, whereas a variant affecting His-96 binds less Ni and abolishes
UreE’s capacity to activate urease (104). These results are easily rationalized by
the crystal structures of Cu-bound K. aerogenes H144*UreE (108) and Zn-bound
B. pasteurii UreE (109). (Figure 1.6) The overall structures are nearly identical,
but contain three and one metal-binding sites, respectively. Both proteins bind a
metal at the dimer interface using the symmetric pair of critical His-96 residues in
the K. aerogenes protein or the pair of His-100 residues for B. pasteun'i UreE.
This metal site is essential for UreE’s function in urease activation. In addition,
each subunit of K. aerogenes UreE binds a metal at sites involving His-110 and
His-112, residues that are substituted with other side chains in the B. pasteurii
protein.

The UreE crystal structures also reveal the presence of two distinct
domains in the proteins. The metal-binding domains, located in the C-temiinal
half of each molecule, resemble the structure of the yeast copper
metallochaperone Atx1 (110). The N-terminal domains have structural similarities

to a domain of the yeast Hsp40 molecular Chaperone Sis1 (111), suggesting that

28

this domain may be involved in molecular recognition and binding to other urease
accessory proteins and/or urease apoprotein. Arguing against this conclusion are
results from studies involving a construct that produced only the metal-binding
domain of K. aerogenes UreE (residues 70-143); the single domain of UreE is
capable of delivering Ni to the urease apoprotein and the N-terminal domain is
not required (112).

Complicating the B. pasteurii UreE structure described above, the
crystallization conditions promote oligomerization of the protein to form a dimer of
dimers (02);» in which all four His100 side chains serve as ligands to a single Zn
(109). The tendency of B. pasteurfi UreE to aggregate was further examined by
protein NMR and equilibrium dialysis approaches (113). Those studies showed
that the tetramer form is favored only at high protein concentrations and provided
evidence for a second Ni-binding site in the C-terminal region of the dimeric form
of UreE, which probably binds a total of 3 Ni ions.

A final comment about urease metallochaperones focuses on the situation
in H. pylori where HypA and HypB, normally associated with hydrogenase
activation, are also required for urease activity. Deletions of either gene encoding
these proteins results in cells with very low urease activity; however, the urease
activity can be restored by addition of excess Ni (30, 52). Thus, it is possible that
HypA, HypB, or a complex of these proteins function as a metallochaperone and

assists in urease activation. These proteins are discussed further below.

29

 

Figure 1.6. K. aerogenes UreE (upper structure, PDB Code 1GMU) and B.
pasteurii UreE (PDB Code 1EAR). The three copper ions coordinated by K.
aerogenes UreE are shown as brown spheres and a zinc ion is shown in gray at
the center of the B. subfilis dimer.

3.2.2. Urease Molecular Chaperones: UreD, UreF and UreG

Structural studies have revealed that the Ni active site of urease is buried
within the enzyme (86), and this site is also relatively inaccessible in the
apoprotein (114). These results are consistent with the need for one or more
urease-specific molecular chaperone(s) to alter the urease protein conformation
and allow Ni to gain access to the active site. From studies with the K. aerogenes
urease system, three proteins, each of which is required for in vivo enzyme
activation, are proposed (1, 94) to act together to fulfill this role: UreD, UreF and
UreG.

As expected of molecular Chaperone proteins, each of the UreD, UreF,
and UreG accessory proteins are found in complexes that include the urease
apoprotein. Thus, UreD-, UreDF-, and UreDFG-urease apoprotein complexes
have been described (115-117). These complexes possess distinct properties
when compared to those of the urease apoprotein alone, especially with regard
to their activation properties. Approximately 15% of the apoprotein is activated in
vitro by addition of 100 pM Ni and 100 mM bicarbonate (needed to carbamylate
the active site Lys) (118, 119). In contrast, about 30% of the UreD-urease
apoprotein is activated by these conditions, demonstrating that UreD directly
enhances this process (115). Furthermore, the UreDF-urease apoprotein is
activated to the same extent by using nearly 1000-fold lower concentrations of
bicarbonate, and activation of this complex is resistant to the detrimental effects
of high concentrations of Ni compared to the apoprotein alone or to UreD-urease

apoprotein (116). A UreDFG-urease apoprotein complex forms upon addition of

31

UreG to UreDF-urease apoprotein (95) and is normally present in cells
expressing the intact K. aerogenes urease gene cluster (117). Significantly, this
species exhibits GTP-dependent urease activation—shown by mutagenesis
studies to be associated with the nucleotide-binding (P-loop) motif located within
UreG (95). Urease activation is not achieved with a non-hydrolyzable analog of
GTP. When the UreE metallochaperone is provided to this complex along with
GTP and near-physiological levels of Ni plus bicarbonate, fully active urease is
generated (97). Three of the urease apoprotein species were probed by a
chemical cross-linking/proteolysis/mass spectrometric approach to examine the
sites of binding of UreD and UreF to urease (120). Additional evidence from
these studies suggests that UreF interacts with UreD-urease apoprotein to give
rise to a conformational change within urease that may enhance access of Ni to
its ligand-binding residues. Finally, we note that a UreDFG complex is generated
in the absence of the structural proteins, and this species was enriched by
binding to an ATP-linked agarose resin (121). Further structural and mechanistic
studies are required to better define the individual roles of UreD, UreF, and UreG
within the heterotrimeric molecular Chaperone that couples GTP hydrolysis to
urease activation.

In addition to participating as a component of the urease molecular
chaperone, UreG has been studied in its purified form. The K. aerogenes protein
is a monomer of 21.9-kDa that, despite having a P-loop motif, fails to bind or
hydrolyze GTP (121). In contrast, UreG from B. pasteun'i, which is over 50%

identical to the K. aerogenes protein, is dimeric and has weak GTPase activity

32

(122). NMR studies of the B. pasteurii protein suggest that it is intrinsically
disordered; however, this UreG was purified from a heterologous overproduction
system and required urea dissolution of inclusion bodies, so the disorder may be
artifactual. Significantly, the purified B. pasteurii UreG binds 2 Zn per dimer (K,
42 pM) or binds 4 Ni per dimer with weak affinity (K, 360 pM).

Two other potential molecular Chaperones for urease exist in H. pylori.
First, is the GroES-homolog called HspA (123). This heat shock protein
possesses an N-tenninal domain resembling the broad specificity molecular
Chaperones, but the protein additionally contains a 27 amino acid extension that
is rich in His. Not unexpectedly, HspA binds 2 Ni with high specificity.
Significantly, when hspA is co-expressed with the H. pylori urease genes in E.
coli, urease activity is enhanced 4-fold in accordance with a possible urease-
specific function (123). The second example of a possible H. pylori-specific
molecular chaperone involves the typical hydrogenase accessory protein HypB
which is required for both urease and hydrogenase activity in this organism.
Since HypB is suggested to function as a molecular chaperone of hydrogenase,
it may also serve this role in urease activation in H. pylori. Alternatively, HypB
along with HypA may possess a metallochaperone role in urease activation in

this microorganism.

33

CONCLUSIONS AND REMAINING QUESTIONS

Accessory proteins are clearly shown to play critical roles in the biosynthesis
of several Ni-containing enzymes. Metallochaperones often are used to deliver Ni
to the target apoprotein and nucleotide-dependent molecular chaperones can
have a role in altering the apoprotein conformation to allow access by the metal
ion. Indeed, the two functions are usually closely linked. Even when such
proteins have been identified, their precise mechanisms of action remain unclear.
For example, it is unknown how Ni becomes bound to metallochaperones and
how the Ni is delivered to the target protein. Similarly, the mechanism by which
nucleotide hydrolysis is coupled to Ni insertion remains a mystery. Furthermore,
these two themes are not universal among all Ni enzymes, as exemplified by Ni-
dependent glyoxylase and aci-reductone dioxygenase, for which no accessory
proteins have been observed; on the other hand, it remains possible that future
investigations will reveal the existence of such accessory proteins for these
cases. Finally, it is important to note that exceptions to the metallocenter
assembly mechanism exist within the particular enzyme systems. For example,
Bacillus subtilis synthesizes sufficient levels of Ni-containing urease to allow for
growth on urea as sole nitrogen source, even though its genome lacks
homologues to ureDEFG (124). The mechanism by which this organism
generates active urease in the apparent absence of any accessory protein
remains unknown.

This thesis aims to shed light on the activation of Klebsiella aerogenes urease

by focusing on selected activation complexes and the accessory protein UreG. In

34

chapter 2, I present structural studies of urease activation complexes (UreABC-
UreD)3 and (UreABC-UreDF)3 performed by small angle X-ray scattering,
flexibility analysis, and site-directed mutagenesis in a combined effort of the
laboratories of Dr. William Heller (Oak Ridge National Laboratory), Dr. Leslie
Kuhn (Michigan State University) and us. Combining the three methods we were
able to propose that a conformational change in UreB would provide access to
the active site of urease, facilitating metallocenter assembly. In chapter 3 I
describe the characterization of a biotinylated form of the accessory protein UreG
and the effect on urease activity caused by single mutations in conserved
residues of UreG. Finally, in chapter 4, I present general conclusions and outline

possible directions for future research.

35

REFERENCES

1.

Mulrooney, S. B., and Hausinger, R. P. (2003) "Nickel uptake and
utilization by microorganisms" FEMS Microbial Rev 27, 239-261.

Hausinger, R. P. (1993) Biochemistry of Nickel, Plenum Publishing, New
York.

Eitinger, T., and Mandrand-Berthelot, M.-A. (2000) "Nickel transport
systems in microorganisms" Arch Microbial 173, 1-9.

Rodionov, D. A., Hebbeln, P., Gelfand, M. S., and Eitinger, T. (2006)
"Comparative and functional genomic analysis of prokaryotic nickel and
cobalt uptake transporters: evidence for a novel group of ATP-binding
cassette transporters" J Bacterial 188, 317-327.

Navarro, C., Wu, L.-F., and Mandrand-Berthelot, M.-A. (1993) "The nik
operon of Escherichia coli encodes a periplasmic binding-protein-
dependent transport system for nickel" Molec Microbial 9, 1181-1191.

de Pina, K., Navarro, C., McWalter, L., Boxer, D. H., Price, N. C., Kelly, S.
M., Mandrand-Berthelat, M.-A., and Wu, L.-F. (1995) "Purification and
characterization of the periplasmic nickel-binding protein NikA of
Escherichia coli K12" Eur J Biochem 227, 857-865.

Heddle, J., Scott, D. J., Unzai, S., Park, S.—Y., and Tame, J. R. H. (2003)
"Crystal structures of the liganded and unliganded nickel-binding protein
NikA from Escherichia coli" J Biol Chem 278, 50322-50329.

Cherrier, M. V., Martin, L., Cavazza, C., Jacquamet, L., Lemaire, D.,
Gaillard, J., and Fontecilla-Camps, J. C. (2005) "Crystallographic and
spectroscopic evidence for high affinity binding of FeEDTA(H20)' to the
periplasmic nickel transporter NikA" J Am Chem Soc 127, 10075-10082.

Allan, C. 8., Wu, L.-F., Cu, 2., Choudhury, S. B., Al-Mjeni, F., Shanna, M.
L., Mandrand-Berthelot, M.-A., and Maroney, M. J. (1998) "An X-ray
absorption spectroscopic structural investigation of the nickel site in
Escherichia coli NikA protein" Inorg Chem 37, 5952-5955.

36

10.

11.

12.

13.

14.

15.

16.

17.

18.

de Pina, K., Desjardin, V., Mandrand-Berthelot, M.-A., Giordano, G., and
Wu, L.-F. (1999) "Isolation and characterization of the nikR gene encOding
a nickel-responsive regulator in Escherichia coli“ J Bacterial 181, 670-674.

Eitinger, T., and Friedrich, B. (1991) "Cloning, nucleotide sequence, and
heterologous expression of the high-affinity nickel transport gene from
Alcaligenes eutrophus" J Biol Chem 266, 3222-3227.

Eitinger, T., Degen, 0., Bbhnke, U., and Miiller, M. (2000) "Nic1p, a
relative of bacterial transition metal perrneases in Schizosaccharomyces
pombe, provides nickel ion for urease biosynthesis" J Biol Chem 275,
18029-18033.

Hartl, F. U., and Hayer-Hartl, M. (2002) "Protein folding - Molecular
chaperones in the cytosol: from nascent chain to folded protein" Science
295, 1852-1858.

Wittung-Stafshede, P. (2002) "Role of cofactors in protein folding" Acc
Chem Res 35, 201-208.

Yan, S. 2., Beeler, J. A., Chen, Y., Shelton, R. K., and Tang, W. J. (2001)
"The regulation of type 7 adenylyl cyclase by its C1b region and
Escherichia coli peptidylprolyl isomerase, SlyD" J Biol Chem 276, 8500-
8506.

Satumba, W. J., and Mossing, M. C. (2002) "Folding and assembly of
lambda Cro repressor dimers are kinetically limited by proline
isomerizatian" Biochemistry 41, 14216-14224.

Scholz, C., Schaarschmidt, P., Engel, A. M., Andres, H., Schmitt, U.,
Faatz, E., Balbach, J., and Schmid, F. X. (2005) "Functional solubilization
of aggregation-prone HIV envelope proteins by covalent fusion with
chaperone modules" J Mol Biol 345, 1229-1241.

Butland, G., Peregrin-Alvarez, J. M., Li, J.-G., Yang, W., Yang, X.,
Canadien, V., Starastine, A., Richards, D., Beattie, B., Kragan, N., Davey,
M., Parkinson, J., Greenblatt, J., and Emili, A. (2005) "Interaction network
containing conserved and essential protein complexes in Escherichia coli“
Nature 433, 531-537.

37

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Rodrigue, A., Batia, N., M'Liller, M., Fayet, O., Bbhm, R., Mandrand-
Berthelat, M.-A., and Wu, L.-F. (1996) "Involvement of the GroE
chaperonins in the nickel-dependent anaerobic biosynthesis of NiFe-
hydrogenases of Escherichia coli" J Bacterial 178, 4453-4460.

Vignais, P. M., and Colbeau, A. (2004) "Molecular biology of microbial
hydrogenases" Curr Issues Molec Biol 6, 159-188.

Vignais, P. M., Billaud, B., and Meyer, J. (2001) "Classification and
phylogeny of hydrogenases" FEMS Microbial Rev 25, 455-501.

Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E. C., Frey, M., and
FontecilIa-Camps, J. C. (1995) "Crystal structure of the nickel-iron
hydrogenase from Desulfavibria gigas" Nature (London) 373, 580-587.

Garcin, E., Vernede, X., Hatchikian, E. C., Volbeda, A., Frey, M., and
FonteciIIa-Camps, J. C. (1999) "The crystal structure of a reduced
[NiFeSe] hydrogenase provides an image of the activated catalytic center"
Structure 7, 557-566.

Volbeda, A., Garcin, E., Piras, C., de Lacey, A. L., Fernandez, V. M.,
Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1996) "Structure
of the [NiFe] hydrogenase active site: evidence for biologically uncommon
Fe ligands" J Am Chem Soc 118, 12989-12996.

Lutz, A., Jacobi, A., Schlensag, V., Bbhm, R., Sawers, G., and Back, A.
(1991) "Molecular characterization of an operon (hyp) necessary for the
activity of the three hydrogenase isaenzymes in Escherichia coli' Molec
Microbial 5, 123-135.

Blakesch, M., Paschos, A., Theodoratau, E., Bauer, A., Hube, M., Huth,
S., and Back, A. (2002) "Metal insertion into NiFe-hydragenases" Biochem
Soc Trans 30, 674-680.

Casalat, L., and Rausset, M. (2001) "Maturation of [NiFe] hydrogenases"
Trends Microbial 9, 228-237.

Blakesch, M., Albracht, S. P. J., Matzanke, B. F., Drapal, N., Jacobi, A.,
and Bock, A. (2004) "The complex between hydrogenase-maturation

38

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

proteins Hpr and Hpr is an intermediate in the supply of cyanide to the
active site iron of [NiFe]-hydrogenases" J Mol Biol 344, 155-167.

Blakesch, M., and Back, A. (2002) "Maturation of [NiFe]-hydrogenases in
Escherichia coli: the Hpr cycle" J Mal Biol 324, 287-296.

Mehta, N. 8., Olson, J. W., and Maier, R. J. (2003) "Characterization of
Helicobacter pylori nickel metabolism accessory proteins needed for
maturation of both urease and hydrogenase" J Bacterial 185, 726-734.

Atanassava, A., and Zamble, D. B. (2005) "Escherichia coli HypA is a zinc
metalloprotein with a weak affinity for nickel" J Bacterial 187, 4689-4697.

Blakesch, M., Rohrmoser, M., Rode, S., and Back, A. (2004) "Hbe, a
zinc-containing protein involved in NiFe hydrogenase biosynthesis" J
Bacterial 186, 2603-261 1.

Maier, T., Jacobi, A., Sauter, M., and Bbck, A. (1993) "The product of the
hypB gene, which is required for nickel incorporation into hydrogenases, is
a novel guanine nucleotide-binding protein" J Bacterial 175, 630-635.

Maier, T., Lattspeich, F ., and Back, A. (1995) "GTP hydrolysis by HypB is
essential for nickel insertion into hydrogenases of Escherichia cali' Eur J
Biochem 230, 133-138.

Fu, C., Olson, J. W., and Maier, R. J. (1995) "HypB protein of
Bradyrhizobium japonicum is a metal-binding GTPase capable of binding
18 divalent nickel ions per dimer" Prac Natl Acad Sci USA 92, 2333-2337.

Rey, L., Imperial, J., Palacias, J.-M., and Ruiz-Argtiesa, T. (1994)
"Purification of Rhizobium leguminasarum HypB, a nickel-binding protein
required for hydrogenase synthesis" J Bacterial 1 76, 6066-6073.

Olson, J. W., and Maier, R. J. (2000) "Dual roles of Bradyrhizobium
japonicum nickelin protein in nickel storage and GTP-dependent Ni
mobilization" J Bacterial 182, 1702-1705.

Olson, J. W., Fu, C., and Maier, R. J. (1997) "The HypB protein from
Bradyrhizobium japonicum can store nickel and is required for the nickel-

39

39.

40.

41.

42.

43.

44.

45.

46.

dependent transcriptional regulation of hydrogenase" Molec Microbial 24,
1 19-128.

Leach, M. R., Sandal, 8., Sun, H., and Zamble, D. B. (2005) "Metal
binding activity of the Escherichia coli hydrogenase maturation factor
HypB" Biochemistry 44, 12229-12238.

Roof, W. D., Horne, S. M., Young, K. D., and Young, R. (1994) "SlyD, a
host gene required for PhiX174 lysis, is related to the FK506-binding
protein family of peptidyl-prolyl cis-trans-isomerases" J Biol Chem 269,
2902-2910.

Hottenrott, S., Schumann, T., Pluckthun, A., Fischer, G., and Rahfeld, J.
U. (1997) "The Escherichia coli SlyD is a metal ion-regulated peptidyl-
prolyl cis/trans-isamerase" J Biol Chem 272, 15697-15701.

Roche, E. D., and Sauer, R. T. (2001) "Identification of endogenous SsrA-
tagged proteins reveals tagging at positions corresponding to stop
codans" J Biol Chem 276, 28509-28515.

Finzi, A., Clautier, J., and Cohen, E. A. (2003) "Two-step purification of
His-tagged Nef protein in native condition using heparin and immobilized
metal ion affinity chromatographies" J Viral Methods 111, 69-73.

Mukherjee, S., Shukla, A., and Guptasarrna, P. (2003) "Single-step
purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase
(PPI), from cytoplasmic extracts of Escherichia coli“ Biatechnal Appl
Biochem 37, 183-186.

Zhang, J. W., Butland, G., Greenblatt, J. F., Emili, A., and Zamble, D. B.
(2005) "A role for SlyD in the Escherichia coli hydrogenase biosynthetic
pathway" J Biol Chem 280.

Blakesch, M., Magalon, A., and Back, A. (2001) "Interplay between the
specific chaperone-like proteins HybG and Hpr in maturation of
hydrogenases 1, 2, and 3 from Escherichia coli" J Bacterial 183, 2817-
2822.

40

47.

48.

49.

50.

51.

52.

53.

55.

56.

Drapal, N., and Back, A. (1998) "Interaction of the hydrogenase accessory
protein Hpr with Hch, the large subunit of Escherichia coli hydrogenase
3 during enzyme maturation" Biochemistry 37, 2941-2948.

Maier, T., Drapal, N., Thanbichler, M., and Bock, A. (1998) "Stept-tag Il
affinity purification: an approach to study intermediates of metalloenzyme
biosynthesis" Anal Biochem 259, 68-73.

Magalan, A., and Bock, A. (2000) "Analysis of the Hpr-Hch complex, a
key intermediate in the assembly of the metal center of Escherichia coli
hydrogenase 3" J Biol Chem 275, 21114-21120.

Hube, M., Blakesch, M., and Back, A. (2002) "Network of hydrogenase
maturation in Escherichia coli: role of accessory proteins HypA and Hbe"
J Bacterial 184, 3879-3885.

Theodoratau, E., Paschos, A., Magalon, A., Fritsche, E., Huber, R., and
Back, A. (2000) "Nickel serves as a substrate recognition motif for the
endopeptidase involved in hydrogenase maturation" Eur J Biochem 267,
1995-1999.

Olson, J. W., Mehta, N. S., and Maier, R. J. (2001) "Requirement of nickel
metabolism proteins HypA and HypB for full activity of both hydrogenase
and urease in Helicobacter pylon" Molec Microbial 39, 176.

Lindahl, P. A. (2002) "The Ni-containing carbon monoxide dehydrogenase
family: light at the end of the tunnel?" Biochemistry 41, 2097-21 05.

Dobbek, H., Svetlitchnyi, V., Gremer, L., Huber, R., and Meyer, 0. (2001)
"Crystal structure of a carbon monoxide dehydrogenase reveals a [Ni-4Fe-
5S] cluster" Science 293, 1281 -1 285.

Drennan, C. L., Heo, J., Sintchak, M. D., Schreiter, E., and Ludden, P. W.
(2001) "Life on carbon monoxide: X-ray structure of Rhadaspirillum
rubrum Ni-Fe-S carbon monoxide dehydrogenase" Prac Natl Acad Sci
USA 98, 11973-11978.

Drennan, C. L., Daukov, T. l., and Ragsdale, S. W. (2004) "The
metallaclusters of carbon monoxide dehydrogenase/acetyl-CaA synthase:
a story in pictures" J Biol lnarg Chem 9, 511-515.

41

57.

58.

59.

60.

61.

62.

63.

65.

Daukov, T. |., lverson, T. M., Seravalli, J., Ragsdale, S. W., and Drennan,
C. L. (2002) "A Ni-Fe-Cu center in a bifunctional carbon monoxide
dehydrogenase/acetyI-CoA synthase" Science 298, 567-272.

Darnault, C., Volbeda, A., Kim, E. J., Legrand, P., Vernede, X., Lindahl, P.
A., and FonteciIIa-Camps, J. C. (2003) "NiZn[Fe4S4] and NiNi[Fe4S4]
clusters in closed and open or subunits of acetyl-CoA synthase/carbon
monoxide dehydrogenase" Nature Struct Biol 10, 271-279.

Svetlitchnyi, V., Dobbek, H., Meyer-Klaucke, W., Meins, T., Thiele, B.,
ROmer, P., Huber, R., and Meyer, 0. (2004) "A functional Ni-Ni-[4Fe-48]
cluster in the monomeric acetyl-CoA synthase from Carbaxydathermus
hydrogenoformans" Prac Natl Acad Sci USA 101, 446-451.

Kerby, R. L., Ludden, P. W., and Roberts, G. P. (1997) "In vivo nickel
insertion into carbon monoxide dehydrogenase of Rhadasprillum rubrum:
molecular and physiological characterization of cooCTJ" J Bacterial 179,
2259-2266.

Watt, R. K., and Ludden, P. W. (1998) "The identification, purification and
characterization of CooJ. A nickel-binding protein that is ca-regulated with

the Ni-containing CO dehydrogenase from Rhadaspirillum rubrum" J Biol
Chem 273, 10019-10025.

Jean, W. B., Cheng, J., and Ludden, P. W. (2001) "Purification and
characterization of membrane-associated CooC protein and its functional

role in the insertion of nickel into carbon monoxide dehydrogenase from
Rhadaspirillum rubrum" J Biol Chem 276, 38602-38609.

Lake, H.-K., and Lindahl, P. A. (2003) "Identification and preliminary
characterization of AcsF, a putative Ni-insertase used in the biosynthesis

of acetyl-CoA synthase from Clastridium thennaaceticum" J lnarg
Biochem 93, 33-40.

Thauer, R. K. (1998) "Biochemistry of methanogenesis: a tribute to
Marjory Stephenson" Microbial 144, 2377-2406.

Ennler, U., Grabarse, W., Shima, S., Goubeaud, M., and Thauer, R. K.
(1997) "Crystal structure of methyl-coenzyme M reductase: the key
enzyme of biological methane formation" Science 278, 1457-1462.

42

66.

67.

68.

69.

70.

71.

72.

73.

74.

Shima, S., and Thauer, R. K. (2005) "Methyl-coenzyme M reductase and
anaerobic oxidation of methane in methanotrophic archaea" Curr Opinion
Microbial 8, 643-648.

Krtiger, M., Meyerdierks, A., Glbckner, F. O., Amann, R., Widdel, F.,
Kube, M., Reinhardt, R., Kahnt, J., Becher, R., Thauer, R. K., and Shima,
S. (2003) "A conspicuous nickel protein in microbial mats that oxidize
methane anaerobically" Nature 426, 878-881.

Hallam, S. J., Putnam, N., Preston, C. M., Detter, J. C., Rokhsar, 0.,
Richardson, P. M., and DeLong, E. F. (2004) "Reverse methanogenesis:
testing the hypothesis with environmental genomics" Science 305, 1457-
1462.

Thauer, R. K., and Bonacker, L. G. (1994) "Biosynthesis of coenzyme
F430, a nickel porphinaid involved in methanogenesis" Ciba Foundation
Symposium 180, 210-227.

Wuerges, J., Lee, J.-W., Yim, Y.-l., Kang, S. O., and Carugo, K. D. (2004)
"Crystal structure of nickel-containing superoxide dismutase reveals
another type of active site" Prac Natl Acad Sci USA 101, 8569-8574.

Barondeau, D. P., Kassman, C. J., Bruns, C. K., Tainer, J. A., and Getzoff,
E. D. (2004) "Nickel superoxide dismutase structure and mechanism"
Biochemistry 43, 8038-8047.

Eitinger, T. (2004) "In vivo production of active nickel superoxide
dismutase from Prochlorococcus marinus MIT9313 is dependent on its
cognate peptidase" J Bacterial 186, 7812-7825.

Kim, l.-K., Yim, Y.-l., Kim, Y.-M., Lee, J.-W., Yim, H.-S., and Kang, S.-O.
(2003) "CbiX-hamologous protein (Cbith), a metal-binding protein, from
Streptomyces seoulensis is involved in expression of nickel-containing
superoxide dismutase" FEMS Microbial Lett 228, 21-26.

Clugstan, S. L., Barnard, J. F. J., Kinach, R., Miedema, D., Ruman, R.,
Daub, E., and Honek, J. F. (1998) "Overproduction and characterization of
a dimeric non-zinc glyoxylase I from Escherichia coli: evidence for optimal
activation by nickel ions" Biochemistry 37, 8754-8763.

43

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

He, M. M., Clugstan, S. L., Honek, J. F., and Matthews, B. W. (2000)
"Determination of the structure of Escherichia coli glyoxylase | suggests a
structural basis for differential metal activation" Biochemistry 39, 8719-
8727.

Clugston, S. L., and Honek, J. F. (2000) "Identification of sequences
encoding the detoxification metalloisomerase glyoxylase l in microbial
genomes from several pathogenic organisms" J Molec Eval 50, 491-495.

Dai, Y., Wensink, P. C., and Abeles, R. H. (1999) "One protein, two
enzymes" J Biol Chem 274, 1193-1195.

Pochapsky, T. C., Pochapsky, S. 8., Ju, T., Mo, H., AI-Mjeni, F., and
Maroney, M. J. (2002) "Modeling and experiment yields the structure of
acireductone dioxygenase from Klebsiella pneumoniae" Nature Struct Biol
9, 966-972.

AI-Mjeni, F., Ju, T., Pochapsky, T. C., and Maroney, M. J. (2002) "XAS
investigation of the structure and function of Ni in acireductone
dioxygenase" Biochemistry 41 , 6761 -6769.

Weeks, D. -L., Eskandar, 8., Scott, D. R., and Sachs, G. (2000) "A H‘”-
gated urea channel: the link between Helicobacter pylori urease and
gastric colonization" Science 287, 482-485.

Olson, J. W., and Maier, R. J. (2002) "Molecular hydrogen as an energy
source for Helicobacter pylori" Science 298, 1788-1790.

Gilbert, J. V., Ramakrishna, J., Sunderman, F. W., Jr., Wright, A., and
Plaut, A. G. (1995) "Protein Hpn: cloning and characterization of a
histidine-rich metal-binding polypeptide in Helicobacter pylori and
Helicobacter mustelae" Infect Immun 63, 2682-2688.

Mobley, H. L. T., Garner, R. M., Chippendale, G. R., Gilbert, J. V., Kane,
A. V., and Plaut, A. G. (1999) "Role of Hpn and NixA of Helicobacter pylori
in susceptibility and resistance to bismuth and other metal ions"
Helicobacter 4, 162-169.

Ge, R., Watt, R. M., Sun, X., Tanner, J. A., He, Q.-Y., Huang, J.-D., and
Sun, H. (2006) "Expression and characterization of the histidine-rich

44

85.

86.

87.

88.

89.

90.

91.

92.

protein, Hpn: potential for nickel storage in Helicobacter pylon" Biochem J
393, 285-293.

Hausinger, R. P., and Karplus, P. A. (2001) in Handbook of
Metalloprateins (Wieghardt, K., Huber, R., Paulos, T. L., and
Messerschmidt, A., Eds.) pp 867-879, John Wiley & Sons, Ltd., West
Sussex, UK.

Jabri, E., Carr, M. B., Hausinger, R. P., and Karplus, P. A. (1995) "The
crystal structure of urease from Klebsiella aerogenes" Science 268, 998-
1004.

Pearson, M. A., Michel, L. O., Hausinger, R. P., and Karplus, P. A. (1997)
"Structure of Cys319 variants and acetohydroxamate-inhibited Klebsiella
aerogenes urease" Biochemistry 36, 8164-8172.

Benini, 8., Rypniewski, W. R., Wilson, K. S., Miletti, S., Ciurli, 8., and
Mangani, S. (1999) "A new proposal for urease mechanism based on the
crystal structures of the native and inhibited enzyme from Bacillus
pasteurii: why urea hydrolysis costs two nickels" Structure 7, 205-216.

Ha, N.-C., Oh, S.-T., Sung, J. Y., Cha, K.-A., Lee, M. H., and Oh, B.-H.
(2001) "Supramolecular assembly and acid resistance of Helicobacter
pylori urease" Nature Structure Biology 8, 505-509.

Sheridan, L., Wilmont, C. M., Cromie, K. D., van der Lagt, P., and Phillips,
S. E. V. (2002) "Crystallization and preliminary X-ray structure
determination of jack bean urease with a bound antibody fragment" Acta
Crystallogr D58, 374-376.

Mancrief, M. B. C., Ham, L. G., Jabri, E., Karplus, P. A., and Hausinger, R.
P. (1995) "Urease activity in the crystalline state" Prat Science 4, 2234-
2236.

Pearson, M. A., Park, l.-S., Schaller, R. A., Michel, L. 0., Karplus, P. A.,
and Hausinger, R. P. (2000) "Kinetic and structural characterization of
urease active site variants" Biochemistry 39, 8575-8584.

45

93.

94.

95.

96.

97.

98.

99.

100.

101.

Mulrooney, S. B., and Hausinger, R. P. (1990) "Sequence of the Klebsiella
aerogenes urease genes and evidence for accessory proteins facilitating
nickel incorporation" J Bacterial 172, 5837-5843.

Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y., and
Hausinger, R. P. (1992) "Klebsiella aerogenes urease gene cluster:
sequence of ureD and demonstration that four accessory genes (ureD,
ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis" J
Bacterial 174, 4324-4330.

Soriano, A., and Hausinger, R. P. (1999) "GTP-dependent activation of
urease apoprotein in complex with the UreD, UreF, and UreG accessory
proteins" Prac Natl Acad Sci USA 96, 11140-11144.

Colpas, G. J., and Hausinger, R. P. (2000) "In vivo and in vitro kinetics of
metal transfer by the Klebsiella aerogenes urease nickel
metallochaperone, UreE" J Biol Chem 275, 10731-10737.

Soriano, A., Colpas, G. J., and Hausinger, R. P. (2000) "UreE stimulation
of GTP-dependent urease activation in the UreD-UreF-UreG-urease
apoprotein complex" Biochemistry 39, 12435-12440.

Jones, B. D., and Mobley, H. L. T. (1989) "Proteus mirabilis urease:
nucleotide sequence determination and comparison with jack bean
urease" J Bacterial 171, 6414-6422.

Musiani, F., Zambelli, B., Stola, M., and Ciurli, S. (2004) "Nickel trafficking:
insights into the fold and function of UreE, a urease metallochaperone" J
lnarg Biochem 98, 803-813.

Lee, M. H., Pankratz, H. S., Wang, 8., Scott, R. A., Finnegan, M. G.,
Johnson, M. K., lppolito, J. A., Christiansan, D. W., and Hausinger, R. P.
(1993) "Purification and characterization of Klebsiella aerogenes UreE

protein: a nickel-binding protein that functions in urease metallocenter
assembly" Prat Science 2, 1042-1052.

Ciurli, S., Safarof, N., Miletti, S., Dikiy, A., Christensen, S. K., Kametzky,
K., Bryant, D. A., Vandenberghe, l., Devreese, B., Samyn, B., Remaut, H.,
and Van Beeumen, J. (2002) "Molecular characterization of Bacillus
pasteurii UreE, a metal-binding chaperone for the assembly of the urease
active site" J Biol lnarg Chem 7, 623-631.

46

102.

103.

104.

105.

106.

107.

108.

109.

110.

Colpas, G. J., Brayman, T. G., McCracken, J., Pressler, M. A., Babcock,
G. T., Ming, L.-J., Colangelo, C. M., Scott, R. A., and Hausinger, R. P.
(1998) "Spectroscopic characterization of metal binding by Klebsiella
aerogenes UreE urease accessory protein" J Biol lnarg Chem 3, 150-160.

Brayman, T. G., and Hausinger, R. P. (1996) "Purification,
characterization, and functional analysis of a truncated Klebsiella
aerogenes UreE urease accessory protein lacking the histidine-rich
carboxyl terminus" J Bacterial 1 78, 5410-5416.

Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999)
"Identification of metal-binding residues in the Klebsiella aerogenes
urease nickel metallochaperone, UreE" Biochemistry 38, 4078-4088.

Benoit, S., and Maier, R. J. (2003) "Dependence of Helicobacter pylori
urease activity on the nickel-sequestering ability of the UreE accessory
protein" J Bacterial 185, 4787-4795.

Rain, J.-C., Selig, L., de Reuse, H., Battaglia, V., Reverdy, C., Simon, S.,
Lenzen, G., Petel, F ,,Wojcik J, Schachter, V., Chemama, Y, Labigne,
A., and Legrain, P. (2001) "The protein- protein interaction map of
Helicobacter pylon" Nature 409, 211--215.

Voland, P., Weeks, D. L., Marcus, E. A., Prinz, C., Sachs, G., and Scott,
D. (2003) "Interactions among the seven Helicobacter pylori proteins
encoded by the urease gene cluster" Liver Physiol 284, GQ6-G106.

Song, H. K., Mulrooney, S. B., Huber, R., and Hausinger, R. P. (2001)
"Crystal structure of Klebsiella aerogenes UreE, a nickel-binding
metallochaperone for urease activation" J Biol Chem 276, 49359-49364.

Remaut, H., 2Safarof, N. ,Ciurli, S., and Van Beeumen, J. (2001)"Structural
basis for Ni2 transport and assembly of the urease active site by the
metallo- chaperone UreE from Bacillus pasteurir" J Biol Chem 276, 49365-
49370.

Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl,
R. A., and O'Halloran, T. V. (1999) "Crystal structure of the Atx1
metallochaperone protein at 1.02 A resolution" Structure 7, 605-617.

47

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

Sha, B., Lee, S.-K., and Cyr, D. M. (2000) "The crystal structure of the
peptide-binding fragment from yeast Hsp40 protein Sis1" Structure 8, 799-
807.

Mulrooney, S. 8., Ward, S. K., and Hausinger, R. P. (2005) "Purification
and properties of the Klebsiella aerogenes UreE metal-binding domain, a
functional metallochaperone of urease" J Bacterial 187, 3581-3585.

Won, H.-S., Lee, Y.-H., Kim, J.-H., Shin, l. S., Lee, M. H., and Lee, B.-J.
(2004) "Structural characterization of the nickel-binding properties of
Bacillus pasteurii UreE in solution" J Biol Chem 279, 17466-17472.

Jabri, E., and Karplus, P. A. (1996) "Structures of the Klebsiella aerogenes
urease apoprotein and two active-site mutants" Biochemistry 35, 10616-
10626.

Park, l.-S., Carr, M. B., and Hausinger, R. P. (1994) "In vitro activation of
urease apoprotein and role of UreD as a chaperone required for nickel
metallocenter assembly" Prac Natl Acad Sci USA 91, 3233-3237.

Moncrief, M. B. C., and Hausinger, R. P. (1996) "Purification and
activation properties of UreD-UreF-urease apoprotein complexes" J
Bacterial 178, 5417-5421 .

Park, I.-S., and Hausinger, R. P. (1995) "Evidence for the presence of
urease apoprotein complexes containing UreD, UreF, and UreG in cells
that are competent for in vivo enzyme activation" J Bacterial 177, 1947-
1951.

Park, l.-S., and Hausinger, R. P. (1995) "Requirement of carbon dioxide
for in vitro assembly of the urease nickel metallocenter" Science 267,
1156-1158.

Park, I.-S., and Hausinger, R. P. (1996) "Metal ion interactions with urease
and UreD-urease apoproteins" Biochemistry 35, 5345-5352.

Chang, 2., Kuchar, J., and Hausinger, R. P. (2004) "Chemical crosslinking
and mass spectrometric identification of sites of interaction for UreD,
UreF, and urease" J Biol Chem 279, 15305-15313.

48

121.

122.

123.

124.

Moncrief, M. B. C., and Hausinger, R. P. (1997) "Characterization of UreG,
identification of a UreD-UreF-UreG complex, and evidence suggesting that
a nucleotide-binding site in UreG is required for in viva metallocenter
assembly of Klebsiella aerogenes urease" J Bacterial 179, 4081-4086.

Zambelli, B., Stola, M., Musiani, F., De Vriendt, K., Samyn, B., Devreese,
B., Van Beeumen, J., Dikiy, A., Bryant, D. A., and Ciurli, S. (2005) "UreG,
a chaperone in the urease assembly process, is an intrinsically
unstructured GTPase that specifically binds Zn2"" J Biol Chem 280, 4684-
4695.

Kansau, l., Guillain, F., Thiberge, J.-M., and Labigne, A. (1996) "Nickel
binding and immunological properties of the C-terminal domain of the
Helicobacter pylori GroES homologue (HspA)" Molec Microbial 22, 1013-
1023.

Kim, J. K., Mulrooney, S. B., and Hausinger, R. P. (2005) "Biosynthesis of
active Bacillus subtilis urease in the absence of known urease accessory
proteins" J Bacterial 187, 7150-7154.

49

Chapter 2

The Structure of Urease Activation Complexes Examined by Flexibility
Analysis, Mutagenesis, and Small-Angle X-Ray Scattering Approaches

The computational analysis included in this chapter were performed by Sai
Chetan K. Sukuru, under the supervision of Dr. Leslie A. Kuhn. The small angle
X-ray scattering data collection and analysis was done by Dr. William T. Heller.

50

ABSTRACT

Conformational changes of Klebiella aerogenes urease apoprotein (UreABC)3
induced upon binding of the UreD and UreF accessory proteins were examined
by a combination of flexibility analysis, mutagenesis, and small-angle x-ray
scattering (SAXS). These studies build on prior work reporting a chemical cross—
link between UreB Lys76 and UreC Lys382 in the (UreABC-UreDF)3 complex
that was interpreted in terms of a conformational change involving UreB. ProFlex
analysis of urease provided evidence that the major domain of UreB can move in
a hinge-like motion to account for the cross-linking result. Rigidification of the
UreB hinge region in the G11P variant was found to reduce the extent of urease
activation, in part by decreasing the nickel content of the mutant enzyme, and to
sequester a portion of the urease apoprotein in an activation complex that
includes all of the accessory proteins. SAXS analyses of urease, (UreABC-
UreD)3, and (UreABC-UreDF)3 are most consistent with UreD and UreF binding
near UreB. Notably, improved fits were observed for (UreABC-UreDF)3 models
where UreB is repositioned in line with the predicted conformational change.
Significantly, the predicted structures of (UreABC-UreDF)3 containing the
domain-shifted UreB conformations allow 002 and nickel ions to gain access to

the nascent active site, compatible with a mechanism for urease activation.

51

INTRODUCTION

Urease is a nickel-containing enzyme that hydrolyzes urea (1, 2).
Crystallographic analyses of ureases from bacterial and plant sources (3-7)
reveal a basic trimeric structure with three active sites, each composed of two
nickel ions coordinated by a carbamylated Lys, four His and an Asp. Genetic and
biochemical studies carried out with plants, fungi, and bacteria [reviewed in (8-
10)] have shown that additional genes encoding accessory proteins are required
for proper assembly of the urease metallocenter, with the possible exception of
Bacillus subtilis (11). The current model for urease metallocenter assembly
(Figure 2.1) derives primarily from studies involving expression of the Klebsiella
aerogenes ureDABCEFG gene cluster in Escherichia coli [reviewed in (8, 12)].
The active enzyme possesses three copies of each of three subunits (UreA,
UreB, and UreC of Mr 11,086, 11,695, and 60,304, respectively)(13). Deletions
within ureD, ureE, ureF, or ureG eliminate urease activity due to production of the
inactive (UreABC)3 urease apoprotein (14). Expression of ureDABC produces
(UreABC-UreD)3, with UreD (Mr 29,300) in complex with urease apoprotein (15).
Co-expression of ureF (encoding a protein of M, 25,221) with ureDABC produces
the (UreABC-UreDF)3 complex (16). The soluble protein UreG (Mr 21,943)
reversibly binds to (UreABC-UreDF)3 forming (UreABC-UreDFG)3 (17, 18).
Urease activity is generated by incubating these complexes with high
concentrations of bicarbonate (to supply the 002 needed for Lys carbamylation)
and nickel ions, but the required levels of these additives (100 mM and 100 pM,

respectively) are not physiologically relevant and only a portion of the proteins

52

are activated (19, 20). In contrast, fully active urease is generated with only 100
pM bicarbonate and 20 pM nickel ions using (UreABC-UreDFG)3 plus UreE (Mr
17,558) and GTP (21). UreE is a nickel-binding protein that delivers the metal ion
to the targeted protein (22, 23), and GTP is hydrolyzed by UreG when present in
the (UreABC-UreDFG)3 complex (24). Although UreE is often referred to as a
metallochaperone (25, 26) and UreDFG has been termed a urease-specific
molecular chaperone (9), the mechanism of urease metallocenter assembly has
remained obscure.

The near identity in structure of the (UreABC)3 apoprotein (27) and the
holoenzyme (3) indicate that conformational changes are required to introduce
the metal ions and 002 into the deeply buried nascent active site. Chemical
cross-linking of (UreABC-UreDF)3 (28) identified a cross-link between UreB
Lys76 and UreC Lys382 that provided evidence for a conformational change of
the protein, since UreB Lys76 is positioned far from UreC Lys382 in the
(UreABC)3 crystal structure. Here, we use computational flexibility analysis to
identify a hinge region that allows the main UreB domain to shift to a position that
allows formation of the critical intra-urease cross-link. Furthermore, we show that
one of two amino acid changes affecting this hinge region leads to a large
reduction in urease activation, partly due to decreasing the extent of nickel
incorporation, while also sequestering a large percentage of the urease protein in
a complex with the accessory proteins. Finally, using SAXS methods we obtain
best-fit models of (UreABC-UreD)3 and (UreABC-UreDF)3 that depict UreD and

UreF binding together with UreB at the perimeter of the disk formed by (UreAC)3.

53

Notably, improved fits were observed for (UreABC-UreDF)3 models where UreB
is repositioned in line with the predicted conformational change. These results
are compatible with earlier urease activation studies and suggest that the
combined action of UreD and UreF serves to expose the nascent active site of

urease.

   

£9

(UreABC)3 (UreABC-Urea)3

  
 

 

@ 3 Ni*%Ni+2
Active ‘31 ‘
Urease 3 00

+ sGDP + 3Pi

FIGURE 2.1. Proposed pathway of urease activation. The K. aerogenes UreA,
UreB and UreC urease subunits assemble into the (UreABC)3 apoprotein
(depicted simply as a trimeric species since UreA plus UreB or all three subunits
are fused together in ureases from some sources). UreD, UreF and UreG
sequentially bind to form the (UreABC—UreD)3, (UreABC-UreDF)3, and UreABC-
UreDFG)3 activation complexes. C02 adds to the active site Lys as Ni+ ions are
delivered to (UreABC-UreDFG)3 by the dimeric UreE metallochaperone in a
process that requires GTP hydrolysis, with UreE and (UreDFG)3 being released
from the activated urease.

54

EXPERIMENTAL PROCEDURES

Protein Purification. (UreABC-UreD)3, (UreABC-UreDF)3, and urease
holoenzyme were produced in E. coli DH5a carrying pKAUD2 (15), E. coli DH5a
pKAUD2F+AureG (16), or E. coli HMS174(DE3) carrying pKK17 (25) and purified
as previously described (29). HEDG buffer (25 mM HEPES, pH 7.4, 1 mM EDTA,
1 mM DTT, 1 % glycerol) was used as a final storage buffer unless noted. The
homogeneity of samples was assessed by densitometric analysis (Alphalmager)
of Coomassie-stained gels after sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) (30). The expression level of urease subunits in cell
extracts was assessed by SDS-PAGE followed by electroblotting the sample
onto lmmobilon-P polyvinylidene difluoride membrane, probing with anti-K.
aerogenes urease antibodies (31), and visualizing with anti-rabbit
immunoglobulin G-alkaline phosphatase conjugates. In a similar manner, the
identity of a contaminating band in one sample was examined by doing a
Western blot with anti-K. aerogenes UreE antibodies (32).

Site-Directed Mutagenesis and Activity Assay. Plasmid pKK17 (25)
containing the entire urease gene cluster was cut with BamHI and the smaller of
two fragments (3.3 kbp) containing ureB was ligated into BamHl-restricted
pUC19 (New England BioLabs), producing pUCB. Mutations of ureB were
introduced by PCR using primers 5’- GAA TAT CAC GTI' AAG CCC _C_CA CAG
ATA GCC CTG AAT ACC -3’ and its complement to introduce the UreB G11P
mutation and 5’- CAG ATA GCC CTG AAT ACC C§A CGG GCA ACC TGT CGC

GTG -3’ and its complement for the UreB G18P mutation (in each case the

55

mutated codon is underlined). The PCR reaction (18 cycles of 50 s at 95 °C, 50 s
at 50 °C and 8 min at 72 °C) was performed with 12.5 pL of PfuTurbo® Hotstart
PCR master mix (Stratagene), 10 pM of each primer, and the pUCB plasmid as
template, followed by incubation for 1 h at 37 °C with 0.5 uL of Dpnl. DH5CI cells
were transformed with 5 pL of the digested PCR reaction. Plasmids from putative
clones were purified, sequenced to confirm the mutations, and digested with
BamHI to recover the 3.3-kbp fragments. These fragments were cloned back into
pKK17 to create pKKBG11P and pKKBG18P.

E. coli cells containing pKK17, pKKBG11P, or pKKBG18P were grown in
Luria-Bertani medium containing 1 mM NiCI2 for three h and induced overnight
with 0.1 mM isopropyl-B—D-thiogalactopyranoside. The stationary phase cells
were harvested by centrifugation, sonicated, and clarified by ultracentrifugation.
Cell extracts were tested for expression of the urease genes by denaturing gel
electrophoresis (30) and subjected to protein analyses (33) and urease activity
assays (34) using standard procedures.

Metal Quantification. The nickel content of selected samples was
assessed by using inductively coupled plasma-mass spectrometry at the
University of Georgia Chemical Analysis Laboratory.

Flexibility Analysis. We used the graph theoretic algorithm ProFlex to
analyze the flexibility of urease (Protein Data Bank (PDB) entry 1FWJ). The
program identifies the flexible and rigid regions in a given structure (which bonds
are constrained and which bonds remain free to rotate) based on analysis of

constraints posed by the protein’s network of covalent bonds, hydrogen bands,

56

salt bridges, and hydrophobic interactions (35). ProFlex calculations have been
shown to predict the conformational flexibility of proteins reliably from a single 3D
structure (35-37). The ProFlex code was modified and extended to allow
processing of the very large urease structure (~22000 atoms in the trimer of
trimers).

SAXS Measurements and Analysis. Small-angle X-ray scattering (SAXS)
data were obtained using the ORNL Center for Structural Molecular Biology 4m
SAXS instrument, described previously (38). Sample intensity patterns were
collected for native urease, (UreABC-UreD)3, and (UreABC-UreDF)3 plus
backgrounds consisting of the buffer solution. Protein concentrations were 3.8
mglmL for native urease, 5.4 mglmL for (UreABC-UreD)3, and 2.0 mglmL for
(UreABC-UreDF)3. These low concentrations made it impractical to measure a
concentration series, but also make it unlikely that interparticle interference
effects are significantly influencing the data. Multiple sample runs were averaged
together, which enabled testing for time-dependent aggregation indicative of
radiation damage; none was found. For (UreABC-UreD)3 and (UreABC-UreDF)3,
four 4-hour runs were summed together, while five 4-hour runs were summed
together for the native urease complex. These measurements included runs with
fresh material and runs in which the sample was exposed for an additional 4
hours to check for radiation damage. No artifacts due to radiation damage were
observed. Data were reduced, azimuthally averaged and scaled into absolute

units (1/cm) according to previously published procedures (38) to provide the 1D

57

intensity profile I(q) vs. q, where q = 4nsin(6l)/,l, 20 is the scattering angle from
the direct beam, and ,1 is the wavelength of the X-ray radiation (1.542 A).
Small-angle X-ray scattering analysis and modeling. Data were subjected
to Guinier analysis (39) for the radius of gyration, R9, and for the pair-distance
distribution function P(r). I(q) and P(r) are related through the Fourier transform

shown in Equation 2.1.

cc
P(r) = 2—7125 qu - I(q)- Sin(qr)- dr (2.1)
O
The program GNOM (40) uses an indirect transform method to find P(r) from an
input maximum linear dimension, dmax. The optimum dmax is found by trial and
error, based on the quality of fit to the input data. The P(r) fitting also provides a
secondary measure of the R9, which is the second moment of P(r).

The program ORNL_SAS (41) was employed to compare the scattering
profiles calculated from the urease structure and various models of complexes
against the measured SAXS profiles of the enzyme, (UreABC-UreD)3, and
(UreABC-UreDF)3. To model the (UreABC-UreD)3 and (UreABC-UreDF)3
complexes, ellipsoids were used in place of the unknown structures of UreD and
UreF. The structures of the higher-order complexes were built by placing three
identical ellipsoids with the (UreABC)3 structure and using the same three-fold
symmetry axis around which the trimer of trimers is formed. The translation
coordinates were chosen randomly from a range of values that made it possible

to produce complexes that extended beyond the experimentally determined dmax.

58

To ensure the proper volume for the added proteins, two of the ellipsoidal
semiaxes were randomly chosen from a range of 10 A to 35 A, and the third was
initially picked to produce the correct expected volume based on the amino acid
sequence of the subunit. In the event that the third semiaxis was found to be less
than 10 A, a new set of semiaxes was generated. The ellipsoids were placed
around the (UreABC)3 structure and the volumes of the ellipsoids that did not
overlap with either the (UreABC)3, or the set of UreD ellipsoids in the case of
(UreABC-UreDF)3, were determined. If the amount of overlapping volume
exceeded 1% of the correct ellipsoidal volume, the ellipsoidal semiaxes were
scaled to provide the correct volume. As the specific overlap region with the other
structures changes as the semiaxes are scaled, an iterative process was
employed until the volume of the overlap regions was less than 1% of the correct
volume. Only the portions of the ellipsoid that did not overlap were retained for
the intensity calculations. Models found to have Rg values consistent with the
experimental data were input into ORNL_SAS for comparison against the
experimental data. ORNL_SAS was configured to treat the density of the
scattering particle as uniform because no atomic-resolution structures are
available for UreD and UreF. A 3 A thick hydration layer, assumed to be 10 %
more dense than the surrounding solution, was used for the ORNL_SAS intensity
calculation. The thickness and density of the hydration layer were not parameters
in the data fitting.

The quality of the fit of the model intensity profiles to the experimental data

was evaluated using the reduced 38 parameter, defined in Equation 2.2.

59

(2.2)

 

 

2
j N; 07(4)
ENLPIS ‘Nf J pm
J

1,: I Z Z (,,(q)_,,,,(q))2

N is the number of data points modelled against in the measured intensity

j, pls

Ij(q). a'J-(q) is the experimental uncertainty in the measured intensity I I(q).
N f is the number of degrees of freedom, and was 2, which accounts for the

scaling of the model intensity profile to the data input into ORNL_SAS.

ORNL_SAS, being a general intensity calculator (41), does not have a

mechanism to account for the ellipsoidal structural parameters in N f. The

number of data points is a great deal larger than the number of degrees of
freedom in any of the models tested, so the impact on X2 is relatively small.
Additionally, each model is tested relative to models generated with the same
number of free parameters, so the relative comparisons are not affected. In order
to judge the range of structures that fit the experimental data collected for
(UreABC-UreD)3 and (UreABC-UreDF)3, the best 25 models found were
maintained in an ordered list that was updated as better models were found, in a
manner similar to previous work (42), making it possible to judge the

reproducibility of the modeling.

60

RESULTS

Flexibility Analysis of Urease. ProFlex, the software designed to analyze
flexibility of proteins (35), was used to examine the flexibility within the native
enzyme trimer of trimers (PDB entry 1FWJ; Figure 2.2 and Figure 2.3 top panels),
identifying a total of ~3100 hydrogen bonds and ~1500 hydrophobic interactions.
The regions of the protein defined as rigid or flexible were found to vary little with
the choice of hydrogen-bond energy cutoff in ProFlex (between -1 and -2
kcal/mol), defining the set of hydrogen bonds and salt bridges incorporated in the
network. In the crystal structure of urease, UreB is anchored by six N-temninal
residues that add to the edge of a beta sheet in UreC (Figure 2.2, region 1). A
salt bridge and at least six hydrophobic interactions between UreB residues 2-8
and UreC residues 6-29 reinforce the attachment (Tables 2.1 and 2.2). ProFlex
predicted UreB residues 11-19 to form a flexible hinge (Figure 2.2, region 2;
Tables 2.1 and 2.4) between the N-terminal anchor and the relatively rigid
domain formed by UreB residues 20-101. The latter domain includes polar and
hydrophobic interactions with UreC (Tables 2.5 and 2.6), but these are few in
number compared to the interactions with regions 1 and 2 and consistent with the
possibility of domain movement. The anchored and hinge residues of the N-
terrninal region of UreB (residues 1-19) fit into a groove of the N-terrninal region
of UreC formed by residues C2-C41 (Figure 2.2).

Chemical modification results (28) indicate that UreB Lys76 and UreC
Ly5382 can be cross-linked when in the (UreABC-UreDF)3 species. This requires

bringing their side chains to within 10 A, although they are 50 A apart in the

61

urease crystal structure. Thus, we probed whether the flexibility of UreB residues
11-19 would allow these two Lys residues to move to within cross-linking
distance while maintaining favorable packing between UreB and UreC. In the first
approach, UreB GIy11 and GIy18 were of special interest due to the prevalence
of Gly in flexible regions of proteins; i.e., Gly residues have no constraints on
main-chain bond rotations (CD and W angle torsions) due to the absence of side-
chain induced steric hindrance. The torsion angles of UreB GIy11 and GIy18
were manually changed to reduce the distance between UreB Lys76 and UreC
Lys382 and attain reasonable packing between UreB and UreAC. The resulting
distance between the Co atoms of UreB Lys76 and UreC Ly5382 was 19.8 A,
close enough to allow cross-linking of their side chains. This motion involved a
rotation of +131 degrees in CD and +110 degrees in \P for GIy11, with 7 degree
changes in both (D and W for GIy18, creating UreB conformation 1 (Figure 2.3,
middle panels). In a second approach, we cut the tether at UreB GIy11, docked
UreB Lys76 within cross-linking distance of UreC Lys282 while maintaining good
packing between the subunits, and reconnected the tether. This approach
created UreB conformation 2 (Figure 2.3, bottom panels). A close-up view
highlighting the repositioning of UreB to achieve conformation 2 and allow cross-

linking is depicted in Figure 2.4.

62

FIGURE 2.2. Tether and hinge regions between UreB and UreC from the
crystallographic structure of urease (A) The native urease structure, with ribbons
colored red for UreA, blue for UreB (except for its hinge and tether to UreC
shown in white), and green for UreC. (B) An expanded view of the region
encircled in yellow in panel A. The N-tenninus of UreB (residues 2-8) forms the
terminal strand of a beta sheet with UreC. UreB residues 11-19 together with
UreC residues 2-6 and 13-41 form a flexible linkage between the main domain of
UreB (blue ribbons in panel A) and the disk formed by (UreAC)3 (red and green
ribbons in panel A). Sites relevant to flexibility probing mutations, UreB Pro10,
GIy11 and GIy18, are rendered as beads. (C) The same view as panel B, colored
in terms of ProFlex flexibility analysis of the crystal structure (PDB entry 1FWJ).
The N-terrninus of UreB partitions from a rigid region (colored blue; region 1) to a
flexible hinge (colored gold; region 2) which connects to the globular domain of
UreB (shown in blue ribbons in panel A). The terminus of UreC is highly flexible
(red), whereas residues in UreC that intervene between regions 1 and 2 are
isostatic, or barely rigid, as shown in grey.

63

Region 2

Mutually Flexible

 

Figure 2.3: Two views of (A) the native conformation of urease, (B) UreB
conformation 1 (torsionally adjusted UreB GIy11 and GIy18 residues), and (C)
UreB conformation 2 (severed linker, docked domain, and reconnected linker).
UreA is depicted in red, UreB in yellow, and UreC in green.

65

 

66

 

FIGURE 2.4. Close-up of the repositioning of UreB from its crystallographic
position (dark blue; PDB 1FWJ) to a position (white) in which UreB Lys76 can
cross link with UreC Lys382 (pink CPK spheres), opening access to the active
site. The range of motion of UreB hinge residues resulting in this rotation of UreB
is shown by the series of blue to lighter blue conformations of residues 11-19
between the UreB crystallographic and cross-linked open positions.

67

Table 2.1: Polar Interactions of Region 1 (UreB residues 2 — 8)

 

 

 

 

 

 

 

 

 

 

Residue 1 322:: Residue 2 Acceptor Atom (xiii/1%»
UreB Gly4 N UreC Ala24 0 -2.73
UreC LysZO N UreB His7 0 -3.62
UreB His7 N UreC LysZO 0 -4.58
UreC Ar922 N UreB G|u5 0 -6.52
UreC Ar922 NH1 UreB Glu5 0E2 -8.11

 

 

Table 2.2: Hydrophobic Interactions of Region 1 (UreB residues 2 — 8)

 

 

 

 

 

 

 

 

 

 

Residue 1 Atom 1 Residue 2 Atom 2
UreB Glu5 CG UreC Trp29 CH2
UreB Tyr6 CD1 UreC Val21 CG2
UreB His7 CB UreC Trp29 CZS
UreB Val8 CG1 UreC Arg6 CB
UreB Val8 CG2 UreC Ala10 CB

 

Table 2.3: Polar Interactions of Region 2 (UreB residues 11 — 19)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Residue 1 3223' Residue 2 Afizfim (£27,320
UreC Arg6 N UreB GIy11 O -3.45
UreC lle4 N UreB lle13 0 -4.53
UreB Leu15 N UreC Ser2 0 -4.73
UreB Asn16 N UreC Tyr39 0 -4.87
UreB |Ie13 N UreC Ile4 O -7.18
UreB Arg19 NH2 UreC Glu41 0E2 -7.34
UreB Arg19 NH1 UreC Glu41 0E2 -8.88
Table 2.4: Hydrophobic Interactions of Region 2 (UreB residues 11 — 19)
Residue 1 Atom 1 Residue 2 Atom 2
UreB lle13 CB UreC lle4 CG2
UreB lle13 CG2 UreC Tyr39 CG

 

 

 

 

 

68

 

 

 

 

Table 2.5: Polar Interactions of Region 3 (UreB residues 20 — 101)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Residue 1 3:3: Residue 2 A‘X‘tifim Energy (Kcal/mol)
UreB Hi539 NE2 UreC Glu41 0E2 -1.03
UreB Ar960 NH2 UreC Glu41 0E1 -1.43
UreB ArgSO NE UreC Glu41 0E2 -1.84
UreC Lys49 NZ UreB Gly66 O -2.11
UreB His87 N UreC Pro102 O -2.20
UreB Ala85 N UreC Ile104 O -2.84
UreB Ala89 N UreC Asp103 0 -3.09
UreB His87 ND1 UreC Asp103 OD1 -4.27
UreB His39 NE2 UreC Glu41 0E1 -8.55
UreB ArgBO NH2 UreC Glu41 0E2 -8.98
Table 2.6: Hydrophobic Interactions of Rem 3 (UreB residues 20 — 101)
Residue 1 Atom 1 Residue 2 Atom 2
UreB Tyr40 CD2 UreC Met55 CE
UreB Phe84 CG UreC lle104 CGZ
UreB Phe84 CD1 UreC lle104 CB
UreB Phe84 CE1 UreC lle104 CD1
UreB Phe91 CB UreC G|n59 CG
UreB Phe93 CE1 UreC Met55 CE
UreB Phe93 CE1 UreC Met55 SD
UreB Phe93 CE1 UreC Met55 CG
UreB Phe93 CZ UreC Met55 CG

 

 

 

 

 

 

Both approaches yielded substantially similar placement of UreB at the periphery
of (UreAC)3 due to the strong constraints placed by maintaining the anchoring
interactions of UreB residues 2-10 while meeting the cross-linking distance

between UreB Lys76 and UreC Lys382.

69

Mutagenesis of Hinge Residues. To directly test the importance of putative
UreB hinge region residues GIy11 and GIy18 in urease activation, their codons
were independently modified to encode Pro residues that would restrict hinge
flexibility. Constructs encoding the G11P and G18P variants of UreB were
created and used to substitute for the wild-type sequence in a plasmid containing
the complete urease gene cluster. The mutated plasmids were transformed into
host E. coli cells, and urease overexpression was shown to be comparable in the
control and mutant strains by using Western blots (data not shown). Urease
activity in cell extracts containing the G18P variant of UreB was similar to that for
extracts containing wild-type enzyme; in contrast, extracts containing the G11P
mutant displayed 15-50 % (depending on the preparation) of the activity of the
control strain.

Urease containing UreB G11P was purified from the mutant strain and
subjected to metal analysis. Whereas control enzyme exhibits a specific activity
of 2,200 :l: 200 pmol min'1 (mg protein") and contains 2.1 :I: 0.3 nickel ions per
active site (43), the purified UreB G11P variant protein possessed a specific
activity of approximately 440 umol min‘1 (mg protein“) and only contained 1.67
nickel ions per active site (single determination with an estimated error of <10 %).
For comparison, 2.13 to 1.74 nickel ions per active site were present after
treating (UreABC)3 with the metal ion or nickel plus bicarbonate yielding specific
activities of 0 and 442 umol min'1 (mg protein)‘1 (20); thus, high nickel content
can be associated with inactive protein. These results suggest both a deficiency

in nickel incorporation and formation of a less effective dinuclear site in the

70

 

mutant protein. Significantly, the mutant urease protein was resolved into two
fractions during phenyl-Sepharose chromatography (Figure 2.5). The highly
purified urease analyzed above was obtained by elution with buffer lacking salt,
as in the case of wild-type enzyme. In addition, a nearly inactive urease-
containing fraction was obtained by subsequent washing of the resin with water;
such a second pool of enzyme is not apparent when purifying wild-type urease.
The second pool of urease contained four major contaminating proteins that co-
migrated with UreD (Mr 29,807), UreG (Mr 21,943), UreF (Mr 25,221), and UreE
(Mr 17,558) (note that the peptides do not migrate precisely according to their
known size). A Western blot analysis with anti-UreE antibodies confirmed the
identity of UreE in this sample. The finding of this apparent complex is
compatible with the need for flexibility in the hinge region of UreB to achieve
accessory protein dissociation. The deleterious effects on urease activity, nickel
content, and accessory protein dissociation that come from restricting the motion
of UreB GIy11 by Pro substitution is consistent with the observation that large
changes in main-chain (D and W values of UreB GIy11 are needed to place Lys76
of this subunit within cross-linking distance of Lys382 in UreC. The neighboring
residue, UreB Pro10, already limits the accessible (D angles so the G11P mutant
would severely restrict the conformations available to the hinge. We hypothesize
that the hinge-like motion of UreB relative to UreC upon binding of UreD and

UreF is associated with the opening of the active site for activation.

71

 

 

kDa
97.4 —

 

a” __ — UreC
45.0 — U D

-_- re
31.0 — iUreG
21.5 —— an» _. .—\UreE
14.4 - '

 

FIGURE 2.5. Two pools of the UreB G11P mutant urease resolved by phenyl-
Sepharose chromatography. Molecular weight standards (Std), the purified active
mutant urease (lane 1), and the very low activity complex containing mutant
urease (lane 2) were examined by SDS-PAGE using a 13.5% acrylamide gel and
stained with Coomassie brilliant blue.

Small-Angle X-Ray Scattering Measurements and Analyses. SAXS data
collected for the three complexes studied are shown in Figure 2.6. Instrument
stability issues, primarily due to temperature fluctuations in the facility, caused
the differences in usable minimum q shown in the graph. The inset curves in
Figure 2.6 are the Guinier regions for the three data sets, and correspond to R9
of 32.7 i 2.4 A , 40.3 i 2.3 A, and 50.6 d: 2.5 A for native urease, (UreABC-
UreD)3, and (UreABC-UreDF)3. respectively. In all cases, the Guinier regions are
linear, indicative of monodisperse scattering particles. The P(r) curves derived
from the SAXS data are shown in Figure 2.7. The R9 for urease determined from
the P(r) fitting was 35.7 :t 0.8 A, with a (1mx of 95 :I: 5 A. The values of R9 for the
(UreABC-UreD)3 and (UreABC-UreDF)3 complexes were 44.9 d: 0.7 A and 53.7 :t
1.4 A, respectively. The dmax of the (UreABC-UreD)3 complex was 130 t 8 A,

while that of the (UreABC-UreDF)3 complex was 155 :I: 10 A. The agreement

72

between the Guinier- and GNOM-derived Rg values is reasonable considering
the very different methods of obtaining the values and estimating the
uncertainties.

Models of the complexes. The intensity profile calculated from the wild-
type urease crystal structure (3) using the program ORNL_SAS (41) is shown
with the data in Figure 2.6. The agreement between the measured data and the
simulated profile is excellent, having a X2 of 0.493. The uncertainties in
measured SAXS intensities derive from specific assumptions about the counting
statistics. In cases of relatively low count rates, the error propagation can result
in uncertainties that overestimate the true uncertainty in the measurement,
making it possible to have x2 significantly less than one. An inspection of the
fidelity of the model profile to the data is required to ensure that the quality of the

fit is truly excellent, as is the case here.

73

._ ______'

 

 

 

A 1
510
'C
a) 0
7°10
0
3’,
em‘
(0

C
9
510'2

10'3

0.03 0.05 0.07010 0.25
cI WA)

FIGURE 2.6. I(q) curves derived from the scattering data for urease (I),
(UreABC-UreD)3 (o) and (UreABC-UreDF)3 (A). The lines are the model fits to
the data using the crystal structure of urease (PDB 1FWJ) (solid line), with UreB
Lys11/Lys18 torsionally adjusted to allow cross-linking of UreB Lys76 to UreC
Ly3382 (dashed line), and UreB docked to UreAC from the crystal structure,
allowing cross-linking of UreB Lys76 to UreC Lys382 (dotted line). The curves
have been offset by a multiplicative factor for clarity. The curves have been offset
for clarity, and the region of data covered by the line indicates the range of data
used for the fitting.

74

P(r) (arb. units)

 

 

 

"'l"'l"'|"'l' r'r'rr'irrii‘r‘
0 20 40 60 80 100 120 140 160
r(

FIGURE 2.7. P(r) curves derived from the scattering data for urease (I),
(UreABC-UreD)3 (o), and (UreABC-UreDF)3 (A). To simplify comparison, the
curves have been scaled to have a value of 1.0 at the peak.

Models of (UreABC-UreD)3 were generated by adding UreD ellipsoids to
the wild-type urease structure and to (UreABC)3 with the two alternative UreB
conformations (one from changes in torsional angles of GIy11 and GIy18 in the
hinge and the other from cleaving the tether, docking of the major UreB domain,
and reconnecting the linker). Ellipsoids were used because no structure or
homology model is available for any UreD. In all cases, the overall structures of
the final complexes were very similar. The best models had UreD ellipsoids
added to the vertices of (UreABC)3 near the UreB subunit such that the total
structure has a planar, triangular character, as can be seen in three pairs of
panels in Figure 2.8. The best three model intensity profiles for the three different

starting structures have x2 of 0.218, 0.252 and 0.224 when starting with the

75

Figure 2.8: Four views (two with UreABC in ribbon and two with UreABC in
spacefilling representation) of the best models of (UreABC-UreD)3 generated by
adding ellipsoids for UreD to the (A) native urease conformation, (B) UreB
conformation 1, and (C) UreB conformation 2. UreA is depicted in red, UreB in
yellow, UreC in green, and UreD in purple.

76

 

77

native structure, UreB conformation 1 (torsionally-adjusted), and UreB
conformation 2 (docked), respectively. In all cases, the fits of the profiles to the
data are excellent and suggest that all of the structures are reasonable. It is
important to note that the three models all have the same general shape, which
is the most reliable result of the modeling considering the method of building the
models and the quality of the data. The addition of UreD results in a planar,
triangular structure. The specific details of the interaction of UreD with UreB
cannot be effectively differentiated with the SAXS data and modeling, in spite of
the differences in 38, because the way in which the ellipsoids were allowed to
conform to the surface of the starting structure enabled them to fill space in such
a way that the final structures have the same general shape. Higher quality data
would not have completely eliminated this ambiguity from the modeling results.
Only a reliable high-resolution structure of UreD, which does not exist, would
have made it possible to differentiate between the different UreB models based
on the SAXS data alone. The (UreABC-UreD)3 results are in agreement with
UreD interacting with UreB as suggested by chemical cross-linking (28), but
would require additional flexibility to accommodate the observed cross-linking of
UreD to UreC Lys401.

The best models for (UreABC-UreDF)3 were created by adding ellipsoids
to represent appropriate molecular volumes of UreD and UreF to the (UreABC)3
crystal structure and the two alternative UreB conformations (to allow for cross-
linking of UreB Lys76 with UreC Lys382). As above, no structure or model is

available for UreD; however, a homology model was reported for UreF from

78

 

Bacillus pasteurii (44). Given the high E-value (4.21) from the 3D-PSSM server
and the fact the model does not represent K. aerogenes UreF, we felt justified in
using ellipsoids to represent this protein. The two alternate UreB conformations,
one produced by docking and the other by torsional adjustments in UreB
residues GIy11 and GIy18, are similar, and in fact resulted in similar placements
of UreD and UreF in the best-fitting SAXS models (shown for the docked
conformation in Figure 2.9). The best UreB conformation 1 (torsionally-adjusted)
and UreB conformation 2 (docked) structures fit the scattering data very well and
have x2 of 0.093 and 0.094, respectively, which is slightly better than the x2 of
0.096 observed for the native structure. The fit of the model profiles to the data
are all excellent, so it is not possible to discriminate between the SAXS models
produced from the different UreB models for the reasons provided above. The
overall shape of the complex, which can be reliably extracted from the data, is
very consistent between the three models, having a planar, triangular character
where the additional mass corresponding to UreD and UreF are located near the
vertices, slightly above the plane defined by the rest of the structure. Reliable
high-resolution structures of the two subunits could provide an effective means to
discriminate between the different UreB structures based on the SAXS data
alone, but no such structures exist for either UreD or UreF. The model depicted
in Figure 2.9 appears to build on the models of (UreABC-UreD)3, with the UreD
and UreF ellipsoids positioned pair wise at the vertices of the (UreABC)3
structure. In this case UreB, UreD, and UreF essentially add onto the edge of the

disk formed primarily by the UreC trimer, in which UreA forms the hub (Figure

79

 

 

2.2). These structures are consistent with immunological results that show anti-
UreD antibodies recognize UreD within (UreABC-UreD)3. but not within (UreABC—

UreDF)3, suggesting that UreF partially masks UreD (16).

 

FIGURE 2.9. Predicted positioning of UreD and UreF relative to the
crystallographic structure of (UreABC)3, based on best-fit models to SAXS data.
The best-fit models resulted in packing of UreD and UreF against UreB near a
vertex of the (UreAC)3 disk. A representative example is illustrated. UreA, UreB,
and UreC are rendered in red, yellow, and green ribbons, respectively. UreD and
UreF from SAXS results are rendered as solid ellipsoids colored purple and
magenta, respectively. The non-interpenetrating volumes of the UreD and UreF
ellipsoids accounts for the appropriate molecular weight of each subunit.
DISCUSSION

In this work we combined multi-scale modeling and sparse experimental
constraints to obtain insight into a flexible molecular assembly, the urease
activation complex. In particular, we used flexibility analysis to provide evidence
that the major domain of UreB can move in a hinge-like motion to allow

sufficiently close juxtaposition of UreB Lys76 with UreC Lys382 to form the

80

 

reported chemical cross-link between these residues, as previously hypothesized
(28). The UreB G11P variant, which is likely to rigidify the hinge region, was
shown to lead to reduced levels of urease activation and lower nickel content
while also sequestering a significant portion of the urease apoprotein in an
ineffective activation complex that includes all four of the known K. aerogenes
accessory proteins. These results support the importance of the hinge region in
urease activation, although we cannot exclude alternative explanations to
account for the properties of the mutant protein. SAXS analysis of urease,
(UreABC-UreD)3, and (UreABC-UreDF)3 were used to provide evidence
consistent with UreD and UreF binding near UreB. Notably, the (UreABC-
UreDF)3 data are best fit by models in which UreB occupies an altered
conformation that would account for the previously described cross-linking results
(28). Significantly, the predicted structures of (UreABC-UreDF)3 containing the
alternative UreB conformations provide access to the nascent active site and
may be a critical step in urease activation.

Comparison of the H. pylori urease structure (PDB entry 1E92) with that of
K. aerogenes urease discussed here provides additional support for the
proposed sites of UreD and UreF interaction with UreB, at the periphery of the
(UreAC)3 disk. The H. pylori UreA subunit (corresponding to a fusion of UreA
and UreB in the K. aerogenes enzyme) contains a fold that matches the K.
aerogenes UreB fold, but also contains residues that add to one side of this
shared fold in a similar position to where we predict UreD and UreF bind. A viral

protein (PDB entry 1C5E) also contains this told with an additional domain in the

81

same region as the added domain in H. pylori UreA. The residues buried on the
part of the H. pylori subunit in common with K. aerogenes UreB are the same in
both the H. pylori and viral proteins, suggesting that this region of UreB has

evolved to interact with other domains or proteins.

ACKNOWLEDGMENTS
I would like to thank Scott Mulrooney and Kimberly Anderson for their

assistance.

82

REFERENCES

1.

Hausinger, R. P., and Karplus, P. A. (2001) Urease, in Handbook of
Metalloprateins (Wieghardt, K., Huber, R., Paulos, T. L., and
Messerschmidt, A., Eds.), pp 867-879, John Wiley & Sons, Ltd., West
Sussex, UK.

Ciurli, S., and Mangani, S. (2001) Nickel-containing enzymes, in
Handbook on Metalloprateins (Bertini, l., Sigel, A., and Sigel, H., Eds.), pp
669-708, Marcel Dekker, New York, NY.

Jabri, E., Carr, M. B., Hausinger, R. P., and Karplus, P. A. (1995) The
crystal structure of urease from Klebsiella aerogenes, Science 268, 998-
1004.

Pearson, M. A., Michel, L. O., Hausinger, R. P., and Karplus, P. A. (1997)
Structure of Cys319 variants and acetohydroxamate-inhibited Klebsiella
aerogenes urease, Biochemistry 36, 8164-8172.

Benini, S., Rypniewski, W. R., Wilson, K. S., Miletti, S., Ciurli, S., and
Mangani, S. (1999) A new proposal for urease mechanism based on the
crystal structures of the native and inhibited enzyme from Bacillus
pasteurii: why urea hydrolysis costs two nickels, Structure 7, 205-216.

Ha, N.-C., Oh, S.-T., Sung, J. Y., Cha, K.-A., Lee, M. H., and Oh, B.-H.
(2001) Supramolecular assembly and acid resistance of Helicobacter
pylori urease, Nature Structure Biology 8, 505-509.

Sheridan, L., Wilmont, C. M., Cromie, K. D., van der Logt, P., and Phillips,
S. E. V. (2002) Crystallization and preliminary X-ray structure
determination of jack bean urease with a bound antibody fragment, Acta
Crystallogr. D58, 374-376.

Mulrooney, S. B., and Hausinger, R. P. (2003) Nickel uptake and
utilization by microorganisms, FEMS Microbial. Rev. 27, 239-261.

Quiroz, S., Kim, J. K., Mulrooney, S. B., and Hausinger, R. P. (2007)
Chaperones of nickel metabolism, in Metal Ions in Life Sciences (Sigel, A.,
Sigel, H., and Sigel, R. K. 0., Eds.), pp 519-544, John Wiley & Sons, New
York.

83

10.

11.

12.

13.

14.

15.

16.

17.

18.

Hausinger, R. P., and Zamble, D. B. (2007) Microbial physiology of nickel
and cobalt, in Molecular Microbiology of Heavy Metals (Nies, D. H., and
Silver, 8., Eds), Springer.

Kim, J. K., Mulrooney, S. B., and Hausinger, R. P. (2005) Biosynthesis of
active Bacillus subtilis urease in the absence of known urease accessory
proteins, J. Bacteriol. 187, 71 50-71 54.

Hausinger, R. P., Colpas, G. J., and Soriano, A. (2001) Urease: a
paradigm for protein-assisted metallocenter assembly, ASM News 67, 78-
84.

Mulrooney, S. B., and Hausinger, R. P. (1990) Sequence of the Klebsiella
aerogenes urease genes and evidence for accessory proteins facilitating
nickel incorporation, J. Bacteriol. 172, 5837-5843.

Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y., and Hausinger,
R. P. ( 1992) Klebsiella aerogenes urease gene cluster: sequence of ureD
and demonstration that four accessory genes (ureD, ureE, ureF, and
ureG) are involved in nickel metallocenter biosynthesis, J. Bacteriol. 174,
4324-4330.

Park, l.-S., Carr, M. B., and Hausinger, R. P. (1994) In vitro activation of
urease apoprotein and role of UreD as a chaperone required for nickel
metallocenter assembly, Prac. Natl. Acad. Sci. USA 91, 3233-3237.

Moncrief, M. B. C., and Hausinger, R. P. (1996) Purification and activation
properties of UreD-UreF-urease apoprotein complexes, J. Bacteriol. 178,
5417-5421.

Park, I.-S., and Hausinger, R. P. (1995) Evidence for the presence of
urease apoprotein complexes containing UreD, UreF, and UreG in cells
that are competent for in vivo enzyme activation, J. Bacteriol. 177, 1947-
1951.

Soriano, A., and Hausinger, R. P. (1999) GTP-dependent activation of
urease apoprotein in complex with the UreD, UreF, and UreG accessory
proteins, Prac. Natl. Acad. Sci. USA 96, 11140-11144.

84

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Park, l.-S., and Hausinger, R. P. (1995) Requirement of carbon dioxide for
in vitro assembly of the urease nickel metallocenter, Science 267, 1156-
1 158.

Park, l.-S., and Hausinger, R. P. (1996) Metal ion interactions with urease
and UreD-urease apoproteins, Biochemistry 35, 5345-5352.

Soriano, A., Colpas, G. J., and Hausinger, R. P. (2000) UreE stimulation
of GTP-dependent urease activation in the UreD-UreF-UreG-urease
apoprotein complex, Biochemistry 39, 12435-12440.

Song, H. K., Mulrooney, S. B., Huber, R., and Hausinger, R. P. (2001)
Crystal structure of Klebsiella aerogenes UreE, a nickel-binding
metallochaperone for urease activation, J. Biol. Chem. 276, 49359-49364.

Mulrooney, S. 8, Ward, S. K., and Hausinger, R. P. (2005) Purification
and properties of the Klebsiella aerogenes UreE metal-binding domain, a
functional metallochaperone of urease, J. Bacteriol. 187, 3581-3585.

Moncrief, M. B. C., and Hausinger, R. P. (1997) Characterization of UreG,
identification of a UreD-UreF-UreG complex, and evidence suggesting that
a nucleotide-binding site in UreG is required for in viva metallocenter
assembly of Klebsiella aerogenes urease, J. Bacteriol. 179, 4081-4086.

Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999)
Identification of metal-binding residues in the Klebsiella aerogenes urease
nickel metallochaperone, UreE, Biochemistry 38, 4078-4088.

Musiani, F., Zambelli, B., Stola, M., and Ciurli, S. (2004) Nickel trafficking:
insights into the fold and function of UreE, a urease metallochaperone, J.
lnarg. Biochem. 98, 803-813.

Jabri, E., and Karplus, P. A. (1996) Structures of the Klebsiella aerogenes
urease apoprotein and two active-site mutants, Biochemistry 35, 10616-
10626.

Chang, 2., Kuchar, J., and Hausinger, R. P. (2004) Chemical crosslinking
and mass spectrometric identification of sites of interaction for UreD, UreF,
and urease, J. Biol. Chem. 279, 15305-15313.

85

29.

30.

31.

32.

33.

35.

36.

37.

38.

Todd, M. J., and Hausinger, R. P. (1989) Competitive inhibitors of
Klebsiella aerogenes urease. Mechanisms of interaction with the nickel
active site, J. Biol. Chem. 264, 15835-15842.

Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly
of the head of bacteriophage T4, Nature (London) 227, 680-685.

Mulrooney, S. B., Pankratz, H. S., and Hausinger, R. P. (1989) Regulation
of gene expression and cellular localization of cloned Klebsiella aerogenes
(K. pneumoniae) urease, J. Gen. Microbiol. 135, 1769-1776.

Lee, M. H., Pankratz, H. S., Wang, 8., Scott, R. A., Finnegan, M. G.,
Johnson, M. K., lppolito, J. A., Christiansan, D. W., and Hausinger, R. P.
(1993) Purification and characterization of Klebsiella aerogenes UreE
protein: a nickel-binding protein that functions in urease metallocenter
assembly, Prat. Science 2, 1042-1052.

Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding,
Anal. Biochem. 72, 248-254.

Weatherburn, M. W. (1967) Phenol-hypochlorite reaction for determination
of ammonia, Anal. Chem. 39, 971-974.

Jacobs, D. J., Rader, A. J., Kuhn, L. A., and Thorpe, M. F. (2001) Protein
flexibility predictions using graph theory, Proteins 44, 150-165.

Hespenheide, B. M., Rader, A. J., Thorpe, M. F., and Kuhn, L. A. (2002)
Identifying protein folding cores from the evolution of flexible regions
during unfolding, J. Molec. Graphics Modeling 21, 195-207.

Rader, A. J., Hespenheide, B. M., Kuhn, L. A., and Thorpe, M. F. (2002)
Protein unfolding: rigidity lost, Proc. Natl. Acad. Sci. USA 99, 3540-3545.

Woodward, J. D., Pickel, J. M., Anovitz, L. M., Heller, W. T., and
Rondinone, A. J. (2006) Self-assembled colloidal crystals from ZrO2
nanoparticles, J. Phys. Chem. B 100, 19456-19460.

86

39.

40.

41.

42.

43.

44.

Guinier, A., and Fournet, G. (1955) Small-Angle Scattering of X-rays, John
Wiley & Sons, New York, NY.

Svergun, D. l. (1992) Determination of the regularization parameter in
indirect-transform methods using perceptual criteria, J. Appl. Crystallogr.
25, 495-503.

Tjioe, E., and Heller, W. T. (2007) ORNL_SAS: software for calculation of
small-angle scattering intensities from bio-macromolecular structures, J.
Appl. Crystallogr. 40, 782-785.

Heller, W. T. (2006) Ellstat: Shape modeling for solution small-angle
scattering of proteins and protein complexes with automated statistical
characterization, J. Appl. Crystallogr. 39, 671-675.

Todd, M. J., and Hausinger, R. P. (1987) Purification and characterization
of the nickel-containing multicomponent urease from Klebsiella aerogenes,
J. Biol. Chem. 262, 5963-5967.

Salomone-Stagni, M., Zambelli, B., Musiani, F., and Ciurli, S. (2007) A
model-based proposal for the role of UreF as a GTPase-activating protein
in the urease active site biosynthesis, Proteins 68, 749-761.

87

 

 

Chapter 3

Mutagenesis of the Klebsiella aerogenes UreG Urease Accessory Protein:
Effects on UreG Properties and Urease Activation

 

88

ABSTRACT

UreG is a GTPase required for assembling the nickel-containing active site of
urease. This urease accessory protein was previously purified from Klebsiella
aerogenes, Bacillus subtilis, and Mycobacterium tuberculosis, with pronounced
differences observed in their respective properties. This work describes an
improved purification method for K. aerogenes UreG that utilizes a biotin tag,
where the fusion peptide was shown to not interfere with urease activation.
Although UreG can form a disulfide-linked dimer, we show that the dimer is not
required for the function of UreG in vivo. The monomeric protein binds 2 nickel
ions per molecule (K, = 12.5 pM), whereas the oxidized protein exhibits greatly
reduced metal binding capacity. Several residues were targeted for mutagenesis,
including four (Cys72, His74, Ser111, and Ser115) with possible sequence
similarity to the dinuclear zinc ligands of the structurally characterized HypB
GTPase of Methanacaldococcus jannaschii and others (LysZO, Asp49, Glu68,
Asp80) thought to function in GTPase activity or metal binding. Single and double
substitutions of these UreG amino acids exhibited little effect on nickel binding,
but most of these alterations abolished UreG’s ability to activate urease. The
biotin tag on UreG was used to isolate a novel complex containing urease
apoprotein along with UreD, UreF, UreG, and UreE. In contrast, the D80A variant
form of UreG interacted only with UreE revealing a new heterodimeric species.
These results suggest a critical role for Asp80 in stabilizing the larger activation

complex.

89

INTRODUCTION

Urease, a nickel-containing metalloenzyme found in plants and
microorganisms, catalyzes the hydrolysis of urea to form ammonia and carbamate,
which spontaneously decomposes to carbon dioxide and ammonia (1). The
structure of the enzyme has been characterized for several species (2-5), and in all
cases the dinuclear nickel metallocenters are deeply buried in structural subunits
that exhibit three-fold symmetry. With the possible exception of the protein of
Bacillus subtilis (6), ureases require a series of accessory proteins to assemble
their active sites (7, 8). The proposed urease activation process (depicted in Fig.
3.1) begins with the structural subunits (UreA, UreB and UreC in the case of the
enterobacterium Klebsiella aerogenes, our model system) assembling into the
urease apoprotein (UreABC)3 (9, 10). The UreD, UreF, and UreG accessory
proteins sequentially associate with the apoprotein to form the (UreABC-UreD)3
(11), (UreABC-UreDF)3 (12), and (UreABC-UreDFG)3 (13) activation complexes.
Finally, in a process that requires GTP, CO2, and the metallochaperone UreE
(which specifically delivers the nickel ions for urease activation), the active site is
assembled and the accessory proteins are released from the active enzyme (14,
15).

The precise roles of the UreD, UreF and UreG accessory proteins are not
well understood. K. aerogenes UreD is insoluble when expressed by itself and no
sequence-related proteins have been structurally characterized, thus preventing
a better understanding of its function. UreF from K. aerogenes also is insoluble

when synthesized separately from the other urease gene products; however, a

90

   

(UreABcie (UreABC-Urea)3

,.@,..
L‘ 3N® filinggm

Act' e o (UreABC- -UreDFG)3
IV - -.

Urease

  

+ 3GDP + 3P
Figure 3.1. Proposed urease activation process. Urease apoprotein (UreABC)3 is

synthesized with the nascent active site lacking nickel and carbamylation of
Ly3217. Urease accessory proteins UreD, UreF, and UreG bind the apoprotein in a
sequential manner to form the (UreABC-UreDFG)3 activation complex. Urease
activation requires carbamylation of Ly5217 by C02, provision of nickel ions by the
UreE metallochaperone, and GTP hydrolysis accompanied by the release the
accessory proteins.

UreE-UreF fusion protein is soluble and has been partially characterized (16). A
fold recognition method was used to create a homology model for UreF of
Bacillus pasteurii, and it was proposed to function as a GTPase-activating protein
(17). Purified recombinant UreG proteins (subunit Mr 22,000 - 23,000) of K.
aerogenes, B. pasteurii, and Mycobacterium tuberculosis are soluble and contain
motifs found in GTPases, although the GTPase activity is very low or not
detectable (13, 18, 19). Mutation of LysZO or Thr21 in the GXGKT P-Ioop motif
(one of the GTPase motifs) of the K. aerogenes protein abolishes urease

activation ( 13) and this region is critical to in vitro activation of the (UreABC-

UreDFG)3 complex (15). Whereas the K. aerogenes protein is monomeric (13),

91

the other two UreG proteins are dimeric with the subunits joined by a disulfide
bridge involving Cys68 (B. pasteurir) and probably Cys90 (M. tuberculosis) (19,
20). UreG of B. pasteurii binds two zinc ions per dimer (K, 42 pM) or four nickel
ions per dimer (Kd 360 pM), perhaps utilizing Glu64, Cy568 (i.e., the residue
proposed to participate in a disulfide), and His70 as metal ligands ( 18) although
no experiments were performed to support these assignments. No crystal
structure is available for any UreG; however, the crystal structure of the related
protein HypB from Methanacaldacaccus jannaschii was reported in 2006 (21).
HypB is an accessory protein that participates in the metallocenter assembly of
Ni-Fe hydrogenases (reviewed in (7, 22) and chapter 1). The crystal structure
reveals two types of zinc binding sites: a mononuclear site in each subunit
involving His100 and His104 (corresponding residues are not present in UreG
sequences) and a non-symmetrical dinuclear binding site at the subunit interface
(Fig. 3.2). The metal-binding residues of the dinuclear site in M. jannaschii HypB
(Cy595, His96, and Cys127) most likely correspond to Cys72, His74, and either
Ser111 or Ser115 in K. aerogenes UreG (or Cys68, His70, and Ser107 or Ser111
in the B. pasteurii protein). Of interest, the Escherichia coli HypB sequence
retains the ligands of the dinuclear center (Cys166, His167, and Cys198), but
lacks the His residues associated with the mononuclear site. In addition, the E.
coli protein contains an amino-terminal extension with a CXXCGC motif, not
found in M. jannaschii HypB or in UreG proteins, responsible for high affinity

(sub-picomolar Kd) binding of a nickel ion (23) .

92

 

Figure 3.2. HypB dinuclear zinc site. HypB from M. jannaschii (PDB code 2HF8)
is a dimeric protein (with the individual subunits depicted in yellow and pink) that
forms an asymmetrical dinuclear site coordinated by Cy395, Hi596, and Cys127.
The zinc atoms are shown as cyan spheres and the water molecules are
depicted as red spheres. Nitrogen atoms are colored in blue and sulfur atoms in
orange.

In this chapter, I describe an improved purification procedure for K.
aerogenes UreG that utilizes a biotin tag, I reexamine the quaternary structure of
the protein, and I explore the effects of mutating several UreG residues on the
properties of the protein, the ability to form activation complexes, and urease

activation.

93

EXPERIMENTAL PROCEDURES

Vector Construction, Cell Growth, and Purification of Biotin- Tagged UreG -
The ureG sequence was subcloned into pASK-IBA3plus and pASK-lBA5plus
plasmids (IBA, Germany) to create vectors plBA3+Gb and plBA5+Gb (Table 3.1)
that encode the protein with a biotin tag (denoted UreGb) at the C- or N-terrnini,
respectively. A polymerase chain reaction (PCR) was performed using PfuTurbo®
Hotstart PCR Master Mix (Stratagene, USA) and the primers 5’-TACT GTC CCG
CGG GATG AAC TCT TAT AAA CAC-3’ and 5’-TACT GTC CTG CAG TI'T GCC
AAG CAT GCC TIT-3’. The first primer contains a Sacll restriction site and the
second a Pstl restriction site that were used to subclone the fragment into pASK-
lBA3plus. In a similar manner, the primers 5’-TACT GTC CCG CGG GG AAC TCT
TAT AAA CAC CCG-3’ and 5’- TACT GTC GGA TCC CTA TI'T GCC AAG CAT
GCC-3’, containing restriction sites for Sacll and BamHI respectively, were used to
subclone the fragment into pASK-lBA5plus. The plasmids and PCR products were
digested with the corresponding restriction enzymes and ligated to produce
plasmids plBA3+G and plBA5+G. Isolated colonies of E. coli DH50 were
transformed with the plasmids and grown at 37°C overnight in 10 mL of Luria
broth (LB) media supplemented with 300 pglmL of ampicillin. These cultures
were used to inoculate 1 L of LB media supplemented with 300 ug/mL of
ampicillin. The cultures were grown at 37°C for 4 h and induced overnight with
100 pl of 2 mg/ml anhydrotetracycline. The cells were harvested by centrifugation
and resuspended in 1 mL of buffer W (100 mM Tris/HCI, pH 8.0, containing 150

mM NaCI and 1 mM EDTA) per g of cells and supplemented with 1 mM

94

TABLE 3.1. Plasmids used in this study

 

 

 

 

 

 

 

Plasmid Description ¥ E. coli I Reference
,, , 7 7 strain
pKK17 - K. aerogenes ' JM109 ‘ (24)
- ureDABCEFG gene cluster ; ‘
. . . Inserted "“0 PKK223 3 ;
pKKGb 3 Modified pKK17 encoding DH5a This work
, f UreGb , -_ _ i .. .
pKKGK20Ab, Modified pKKGb encoding I DH5a This work
pKKGD49Ab, ithe K20A, D49A, E68A, a 5
pKKGE68Ab, C72A, H74A, H74C, H74N,
pKKGC72Ab, D80A, S111A, and S115A
pKKGH74Ab, 5 variant forms of UreGb =
pKKGH74Cb, : 5
pKKGH74Nb,
pKKGD80Ab,
pKKGS111Ab, and
..PKK63115AD... , . . ,;. .....
pASK—IBA3plus Plasmid for creating fusion 1 DH5a IBA
proteins with a biotin tag at
Lthe Q-tsrmmus. ..
pASK-IBA5plus j Plasmid forcreating fusion : DH50c ; IBA
proteins with a biotin tag at
the N-tem‘rinus .--. -...z-.-_.-_-_-~
plBA3+Gb ‘ Modified pASK-lBA3pIus to DH5a ‘3 This work
I encode biotin-tagged
.__ LUreGb
plBA5+Gb Modified pASK- |BA5plus to DH50. This work
encode biotin-tagged ..
.. . ..i.UreGb,.. ., , . . .. -
plBA3+GK20Ab, f Modified plBA3+Gb to i BL21 This work
plBA3+GD49Ab, I encode the K20A, D49A, '
plBA3+GE68Ab, E68A C72A, H74A, H74C,
plBA3+GC72Ab, g H74N, S111A, C72AIH74A,
plBA3+GH74Ab, ' C72A/S111A, and
plBA3+GH74Cb, H74A/S111A variants of
plBA3+GH74Nb, 3 UreGb
plBA3+GS111Ab,
plBA3+GC72A/H74Ab,
plBA3+GC72A/S111A,
and i
plBA3+GH74NS111Abi

 

 

95

phenylmethylsulphonyl fluoride (PMSF) before sonification (Branson 450 sonifier,
5 repetitions, each of 2 min, at 3 W output power and 50% duty cycle). The lysate
was centrifuged at 100,000 g at 4°C for 45 min and the supernatant was loaded
onto a 1 ml Strep-Tactin column (IBA, Germany) previously equilibrated in buffer
W. This column has an engineered streptavidin ligand that binds biotin with high
affinity. The protein was eluted with desthiobiotin according to the manufacturer’s
instructions. For comparative studies, native UreG was purified as previously
described (13).

Fractions containing UreGb, UreG, or mutants were analyzed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (25) using gels
prepared with 13.5% acrylamide and stained with Coomassie brilliant blue.
Molecular weight markers were obtained from Bio-Rad (Hercules, CA). Protein
concentrations were determined by using a commercial assay (Bio-Rad, Hercules,
CA) with bovine serum albumin as the standard. In some cases the protein was
loaded onto a preparative Superdex-75 column (65 cm x 1.5 cm diam., Amersham,
USA) equilibrated in 50 mM HEPES buffer, pH 7.4, containing 200 mM NaCI and
chromatographed at 1 lemin in this buffer for further purification.

Site-Directed Mutagenesis - Mutations were generated in plBA3+G by using
overlapping Oligonucleotides containing the desired mutation (see Table 3.2). The
PCR was performed with PfuTurbo® Hotstart PCR Master Mix and the
corresponding Oligonucleotides. The products were digested with Dpnl for one h at

37°C and used to transform chemically competent E. coli DHSo, cells. The

mutations were confirmed by sequencing (Davis sequencing, Davis, CA, USA). The

96

mutated plasmids were purified and used to transform E. coli BL21 Gold competent
cells (Stratagene, USA). Double mutants (C72AlH74A, H74/S111A and
C72AIS111A) were prepared by using as a template a previous mutant. All

mutants were expressed and purified as described for UreGb.

TABLE 3.2. Oligonucleotides used to generate ureG mutations

 

Purpose Sequence

 

UreG mutation D49A 5’ - GAC ATC TAT ACC AAA GAA GCG CAG CGC
ATC CTC ACC GAA - 3’

 

UreG mutation E68A 5’- GAA CGC ATC GTC GGT GTG GCG ACC GGC
GGC TGC CCG CAT- 3’

 

UreG mutatia'n‘GTiA' " 5" GTG GGT GTG GAA Acc GGG GGC G_c__G CCG
GAT ACG GCG ATC CGC GAA g:

 

 

 

UreG mutation HTXA“ ” 5' GAA ACC GGC GGC TGc GCG Gc"___A_ ACG GCG
ATC CGC GAA GAT 3'

 

 

UreG mutation H74C 5' - GAA ACC GGC GGC'TGGGCG TGC ACG'GGG“
ATC CGC GAA GAT - 3'

 

UreG mutation H74N 5'- GAA ACC GGC GGC TGCCCGAATACGGCG”
ATC CGC GAA GAT 3'

 

 

UreG mutation D80A 5’- CAT ACG GCG ATC CGC GAA GCG GCC TCA
ATG AAC CTC GCC- 3’

 

 

 

 

UreG mutation S111A 5'- A GAA AGC GGC GGC GAT AAC CTG _G___CC GCC
ACC TTC AGC CCG GAG CTG- 3'

 

P UreG mutation S115A 5'- AAC CTG AGC GCC ACC TTC GCC CCG GAG
CTG GCG GAT CTG- 3'

 

 

 

Double UreG mutation ‘5' GT0 GGT GTG GAA ACC GGc GGc G__C_G CCG
C72A/H74A GCA ACG GCG ATC CGC GAA 3'

 

 

Circular Dichroism (CD) - Proteins were purified and concentrated to 0.2
mglmL in 15 mM phosphate buffer, pH 7.6, containing 1 mM dithiothreitol (D‘I‘I’).
A 100 pL sample was placed into a Jasco J-710 spectropolarimeter and data
collected between 180 and 300 nm with a 1 cm path length. The data were
analyzed with the DICHROWEB server (26). The best fit was obtained using

CDSSTR and set 4.

97

 

Analytical Gel Filtration Chromatography - Superdex-75 (45 cm x 1.0 cm
diam., Amersham, USA) was used for analytical hydrodynamic radius assays.
The buffer contained 50 mM HEPES, pH 7.4, containing 200 mM NaCl and other
additives as indicated, with the flow at 1 ml/min.

Metal Quantification - The nickel content of freshly purified UreG and
UreGb was assessed by using inductively coupled plasma-mass spectrometry at
the University of Georgia Chemical Analysis Laboratory.

Nickel Binding - Purified proteins were dialyzed overnight against 50 mM
HEPES buffer, pH 7.4, containing 200 mM NaCI, 10 mM EDTA, and 1 mM DTT,
followed by dialysis 4 times against 50 mM HEPES buffer, pH 7.4, containing 200
mM NaCl. The samples were incubated for 30 min at 4°C with different
concentrations of nickel, and centrifuged at 14,000 g in a tabletop centrifuge for
20 min using a Microcon® (Millipore, USA) centrifuge unit with a nominal
molecular weight cut off of 10 kDa. A 100, 50 or 20 uL aliquot of the flow-through
fraction was adjusted to 100 uL (as needed), mixed with 900 pL of 100 pM 4-(2-
pyridylazo)-resorcinol (PAR), and analyzed spectrophotometrically to determine
the amount of metal. The data were plotted and analyzed in Sigma Plot using the
following equation where [Ni] is the concentration of free nickel ion, Bmax is the
maximum number of nickel ions bound per UreG peptide, Nib is the number of
nickel ions bound per UreG and K, is the dissociation constant.

Nib = (Bmax x [MD I (Kd+ [Ni] )

Analysis of Cells Expressing the Urease Operon Encoding the UreGb

Variants - Plasmid pKK17(24), which contains the entire ureDABCEFG urease

98

gene cluster under the control of the lac promoter, was modified to encode UreGb
and its mutant forms by replacing a Psrl/Kpnl fragment. For analysis of urease
activity in cell extracts, a single colony containing the desired plasmid was
inoculated into 1 mL of LB media supplemented with 300 pg/mL of ampicillin and 1
mM NiClz (unless noted) and grown overnight at 37°C with agitation. A 0.5 mL
aliquot of the culture was used to inoculate 50 mL of LB containing 300 ug/mL of
ampicillin and 1 mM NiCl2 (unless noted) and grown for 2.5 h at 37°C with agitation.
lsopropyl B—D-1-thiogalactopyranoside (IPTG) was added to 0.1 mM to induce the
expression of the operon overnight at 37°C with agitation. Cells were harvested by
centrifugation for 10 min at 5,000 g and 4°C and resuspended in 1 mL of 25 mM
HEPES buffer, pH 7.4, for urease activity assays. If the samples were to be used
for pull-down assays, cells were resuspended in 750 pl of buffer W. PMSF was
added to 0.1 mM, the cells were sonicated (Branson 450 sonifier, 5 repetitions,
each of 45 sec, at 1 W output power and 50% duty cycle), and the disrupted cells
were centrifuged 10 min at 4°C and 16,000 gin a microcentrifuge. The cell extracts
were used to test urease activity and perform pull down assays.

Urease Activity Assays - Urease activities were measured by quantifying the
rate of ammonia release from urea by formation of indophenol, which was
monitored at 625 nm (27). One unit of urease activity was defined as the amount of
enzyme required to hydrolyze 1 pmole of urea per min at 37°C. The standard assay
buffer consisted of 50 mM HEPES, pH 7.8, and 50 mM urea.

Pull-Down Assays - Samples (0.5 mL) of cell extracts (from E. coli DH5a

containing pKK17 with the modified versions of ureG) were loaded onto a 0.3 mL

99

Strep-Tactin column (IBA, Germany) equilibrated in buffer W. Proteins were eluted
according to the manufacturer’s instructions and analyzed using 13.5% SDS-
PAGE.

Westem Blot - Proteins resolved by SDS-PAGE were transferred to an
lmmobilon-P polyvinylidene difluoride membrane (Millipore, USA). ExtrAvidin®-
alkaline phosphatase conjugate (1:2500 dilution, Sigma, USA) was used as a probe
to bind to biotin-tagged forms of UreG. BCIP®INBT-Blue Liquid Substrate (Sigma,
USA) was added to develop the color. To detect UreE or urease, the membranes
were incubated for 45 min with anti-UreE lgG (1:10,000 dilution) or anti-urease
antibody (1:5,000 dilution) in TBS buffer (150 mM NaCl, 100 mM Tris, pH 7.4)
containing 1% Tween 20. After washing the membranes four times with TBS,
they were incubated for 45 min with anti-rabbit lgG conjugated to alkaline
phosphatase (Sigma, USA) diluted 30,000 times. The membranes were washed
again and the BCIP®INBT—Blue Liquid Substrate was added to develop the color.

Prestained molecular weight markers were obtained from Bio-Rad (Hercules, CA).

RESULTS

Characterization of Biotin- Tagged UreG (UreGb) - The native form of K.
aerogenes UreG was previously purified from recombinant E. coli cells by using a
series of three different columns (13); however, the tendency of the protein to
elute from ion exchange resins over a large number of fractions led to low overall
yields. To overcome the low yield problem and to facilitate a single-step

purification of UreG variants, we examined a new purification system involving a

100

fusion peptide sequence that becomes biotinylated. This tag was specifically
designed to allow affinity purification without introduction of metal-binding
residues as in the commonly used Hiss tag (28). The ureG sequence was
subcloned into plasmid pASK-lBA3plus and pASK—lBA5plus so as to encode
UreG fused with a biotin-tagged peptide at the C- or N-terrnini, respectively. The
protein derived from the pASK-lBA3plus vector was overproduced in higher
amounts by cells, so this plasmid was selected for further experiments. For
comparative analyses, native UreG also was obtained by using the previously
described protocol (13).

UreGb was highly purified by single—step chromatography on a Strep-
Tactin column, and essentially homogeneous protein was obtained by
subsequent gel filtration chromatography (Fig. 3.3). The biotin-tagged UreGb
possessed nearly the same secondary structure as the native UreG according to
CD measurements (60% a helix, 18% [3 strands, 4% turns, and 18% random coil
for UreGb, versus 65% a helix, 15% B strands, 5% turns and 15% random coil for
native UreG, each with a normalized root mean square deviation of 0.001; Fig.
3.4). Results obtained by size exclusion chromatography were consistent with
UreGb being strictly a monomeric protein (Fig. 3.5), even when 0.5 mM nickel or
zinc ions were added to the buffer (data not shown). This finding contrasts with
the situation for native K. aerogenes UreG which gives rise to features consistent
with both monomeric and dimeric species, although the latter is present in small
amounts (Fig. 3.5). The dimer disappears when the protein is dialyzed overnight

with 1 mM DTT, suggesting the presence of some disulfide-linked subunits. The

101

ratio of dimer to monomer does not change in the presence of nickel or zinc (data
not shown), but it increases after several days of protein storage, consistent with

formation of an oxidation product.

StdExFl'123456

kDa

97.4 —
66.2 —
45.0 _
31.0 '—
21.5 —

14.4 —‘ '

 

Figure 3.3. UreGb purification. UreGb was isolated by use of a Strep-Tactin
affinity column followed by gel filtration chromatography. The purified sample was
subjected to SDS-PAGE analysis followed by staining with Coomassie brilliant
blue. Lanes: Std, standard proteins used as size markers; Ex, cell extracts; FT,
flow through; 1-6, fractions recovered by elution with desthiobiotin.

   

E” §’

E E

E 1 a?

4": i . g

.9- f ‘ .9-

E . E
-30 . , ~ -10 - . . -»
190 210 230 250 190 210 230 250

Wavelength (nm) Wavelength (nm)

Figure 3.4. CD spectra for UreG and UreGb. The proteins were analyzed in the
190-260 nm range at 0.2 mg/ml and 0.1 mg/ml respectively. Panel A shows UreG
spectra and panel B shows UreGb.

102

 

0.030
0.025
0.020
0.015
0.010

 

0.005

Absorbance 280 nm

 

 

 

0.000 R

 

 

-0.005

 

0 10 20 30 40
Elution time (min)

Figure 3.5. Size exclusion profile of native UreG and UreGb. 300 pl of a 2 mglml
(app. 90 pM) solution of UreG or UreGb were loaded onto a 35 ml Superdex-75
column (1.0 x 45 cm). The buffer contained 50 mM HEPES, pH 7.4, 200 mM
NaCl and 0.5 pM NiNOz .The solid line corresponds to UreG and the dashed line
corresponds to UreGb.

Targeting Residues for Mutagenesis — Two criteria were used to select
amino acids for mutagenesis. First, a sequence alignment that included

sequences of UreG and HypB from 30 different organisms published in Pfam-B,

release 4.0 (http://pfamsangeraculd) was used to find highly conserved

 

residues. The high level of identity between UreG proteins of different species
(over 50%; see complete sequence comparisons in (18, 19)) precludes the
identification of critical amino acid residues by simple sequence alignment;
however, UreG sequence comparison with the related protein HypB highlights

fewer amino acids. For example, the P-loop motif (GSGKT at positions 17-21 in

103

K. aerogenes UreG or residues 43-47 of M. jannaschii HypB), the signature motif
for the SlMBl G3E family of GTPases (29) (ESGG at positions 104-107 of UreG
or ENVG at 120-123 of HypB), and the guanine specificity loop (NKT D at
positions 151-154 of UreG or NKID at residues 167-170 of HypB) were
conserved as expected. Second, the crystal structure of M. jannaschii HypB (21)
showed a dinuclear zinc binding site. We aimed to identify the corresponding
amino acids to verify if the same metal binding site exists in UreG. Therefore, the
residues targeted for mutagenesis included: Lys20, previously shown to be a
critical P-loop residue (13) and earlier changed to K20A; Asp49, equivalent to the
Mg+2-coordinating Asp75 in M. jannaschii HypB (21) was changed to D49A;
Glu68, the corresponding residue of which was suggested to be a metal ligand
for the B. pasteurii protein (18), was changed to E68A; Cys72, likely to
correspond to the Cys95 metal ligand at the dinuclear site of M. jannaschii HypB
(21) was changed to C72A; His74, likely to correspond to the Hi596 dinuclear
center ligand of HypB (21) was changed to H74A, H740, and H74N; Asp80,
corresponding to Asp98 of HypB and highly conserved in both proteins, was
changed to D80A, and Ser111 and Ser115, likely to correspond to the Cys127
ligand of the dinuclear site in HypB were changed to S111A and S115A. Three
double mutants were also constructed: C72A/H74A, C72A/S111A, and
H74A/S111A.

Nickel Binding to UreG, UreGb and Variants -—UreG and UreGb contained
no significant levels of bound nickel or zinc ions as freshly purified by the

methods described above (data not shown). The nickel ion-binding properties of

104

UreG, UreGb, and selected mutant proteins were examined by a
centrifugation/PAR reactivity approach (see Experimental Procedures). This
method revealed that the fully reduced and monomeric native form of UreG
bound 2.0 i 0.1 nickel ions per molecule with a Kd of 12.5 i 3.3 uM (Table 3.3).
In contrast, a partially oxidized sample of UreG bound 1 nickel ion per molecule
with a K, of 46.6: 13.6, compatible with thiol group participation in metal binding.
The strictly monomeric UreGb binds 2.0 i 0.1 nickel ions per molecule with a Kd
of 29.9 i 19.1 uM (Table 3.3 and Fig. 3.6). This result is consistent with the biotin
tag leading to decreased metal ion binding affinity as well as prevention of
dimerization, potentially due to interference with a thiol group. The C72A and
S111A single mutants of UreGb bound 1.5 i 0.1 nickel ions per molecule and the
H74A variant binds 1.7 :l: 0.2 nickel ions per molecule, all with lower Kd than
observed for UreGb and comparable to that noted for UreG (Table 3.3 and Fig.
3.6). Similarly, the C72A/S111A and H74A/8111A double mutants of UreGb
exhibited the same properties (Table 3.3). The C72A/H74A UreGb double mutant

was isolated in very low amounts, thus preventing metal-binding analysis.

Table 3.3. Thermodynamics of nickel ion binding to UreG roteins.

 

 

 

 

 

 

 

 

 

 

Protein* Kd Bmax

UreG (monomer) (1) 12.5 i 3.3 2.0 i 0.1
UreG (partial dimer) (3) 46.6 i- 13.6 1.0 i 0.1
UreGb (3) 29.9 i 19.1 2.0 :l: 0.1
UreGC72Ab (3) 15.8 i 3.6 1.5 :l: 0.1
UreGH74Ab (3) 9.2 i 2.3 1.7 :t 0.2
UreGS111Ab (2) 12.5 i 3.6 1.5 :t 0.1
UreGC72A/S111Ab (1) 10.9 i 2.8 1.5 i 0.1
UreGH74A/S111AAb(3) 12.1 1: 4.9 1.6 i 0.1

 

 

 

 

*The numbers in parentheses indicate the number of experiments.

105

 

 

 

 

Ni/protein

 

 

 

0 100 200 300 400 500

Free Ni (pM)
Figure 3.6. Metal binding to UreGb and selected variants. Samples were
incubated with varied concentrations of nickel ions, and portions of the buffers
containing the free metal ion were separated from proteins using a Microcon
centrifuge unit. The nickel ion concentrations of the protein-free solutions were
determined spectrophotometrically using the colorimetric reagent PAR. UreG
samples included: UreGb (closed circles, data fitting in solid line), C72A (open
circles, data fitting in dash line) and H74A (gray squares, data fitting in dots and
dash)

Effect of UreGb Variants on Urease Activity in Cell Extracts —Selected
versions of ureG were expressed as part of the urease operon, and the levels of
the encoded UreGb variants were shown to be indistinguishable when cell
extracts were examined by Western blot (Fig. 3.7). The urease activity measured
in extracts of cells producing UreGb was indistinguishable from that of extracts
from cells containing native UreG (Fig. 3.8). In contrast, the cells producing
K20A, D49A, C72A, H74A, H74C, H74N, D80A, and S111A variants of UreGb
led to nearly undetectable levels of urease activity. Extracts from cells containing

the E68A UreGb variant possessed about 18% of the wild-type level of urease

activity. Finally, the S115A mutant had no effect on urease activation.

106

 

Figure 3.7. Analysis of mutant UreGb levels in cell extracts. Extracts of E. coli
BL21 cells containing derivatives of pKKGb were subjected to SDS-PAGE, the
proteins were transferred to an lmmobilon-P membrane, and phosphatase-
conjugated avidin was used to assess the content of biotin-tagged proteins in the
samples. The lower band likely corresponds to the endogenous carboxyl carrier
protein in the cells, whereas the upper band corresponds to the UreGb variants.
Lanes: Std, prestained molecular weight standards; UreGb variants cell extracts;
-Ni and +Ni correspond to cells containing pKKGb plasmid grown without or with
nickel respectively.

300
250
200
1 50
100
50
0 L 5,;

'0
0&0

 

Ulmg total protein

 

   

vv-quvoevvv-
9Q» our-hone
otéédbssstilgégé

 

Figure 3.8. Urease activity in cell extracts expressing UreGb and its mutants. The
urease activity was examined in extracts of E. coli DH5a cells with the variant
pKKGb plasmids encoding the various forms of biotin-tagged UreG. For
comparison, activity of cells containing pKAU17 (also containing the complete
urease operon) was 198 U/mg (30, 31). The error bars indicate the standard
deviation of at least three independent measurements.

107

Pull-down Assays — A lack of urease activity in extracts of cells containing
altered forms of UreGb could be caused by an inability to form the (UreABC-
UreDFG)3 activation complex. We exploited the biotin tag on UreGb to examine
the ability of the UreGb variants to interact with other proteins to form complexes
in vivo. Cells expressing UreGb from the urease operon were grown with or
without added nickel as a control. When extracts of cells containing the entire
urease operon and encoding UreGb were loaded onto the Strep-Tactin column,
washed, and eluted, several bands were observed by SDS-PAGE in addition to
UreGb (Fig. 3.9). Urease structural subunits were identified by their characteristic
size and by Western blot using anti-urease antibodies (data not shown).
Additional bands migrated at positions expected for the UreD and UreF
accessory proteins. Finally, an extra band was identified as UreE by using anti-
UreE antibodies in a Western blot (Fig. 3.10). This band was shown to be
present in all samples. Of potential interest, UreE was present in smaller
amounts for cultures grown in the absence of added nickel ions than for the
sample grown in its presence, suggesting that the metal-bound form of UreE
preferentially binds to this complex. The sample derived using extracts containing
the D80A UreGb variant possessed very low quantities of the urease structural
subunits and appeared to lack UreD and UreF, yet the band corresponding to
UreE was present. This finding is consistent with a direct UreG-UreE interaction.
The samples obtained for extracts containing the K20A, H74A and S111A UreGb
variants bound weakly to the column, as if the biotin tag was inaccessible in

these UreGb-containing complexes, perhaps indicating that the proteins in the

108

complexes mask or obscure the UreGb carboxyl terminus. In contrast, the
samples containing each of the three His74 UreGb variants exhibited complexes
that bound to the resin, with the H74A UreGb sample appearing identical to the
non-mutated UreGb. It is interesting to note that variant H74C of UreGb led to a
more intense band corresponding to UreE in that gel, as if this mutation led to
binding stabilization of the UreE apoprotein. Finally, the E68A, C72A and H74N
variants of UreGb resulted in complexes that behaved like those for the non-

mutated UreGb.

109

 

 

    

Std E68 C2 D80 -Ni +Ni K20 D49 H74 8111

kDa
97.4 '-
uu — — ”rec
“'0 -‘ UreD
31.0 — ”'09
_‘UreF
21.5 — "\UreE
_. UreB
14.4 -"
'_ UreA
H74 to
kDa
97.4
33.2 —‘
45.0
31.0
21.5
14.4

 

Figure 3.9. Pull-down assays. Extracts of E. coli BL21 cells containing the
various pKKGb plasmids that encode variant forms of UreGb along with all other
urease structural and accessory proteins were chromatographed on Strep-Tactin
columns. The samples that were eluted with desthiobiotin were subjected to
SDS-PAGE and stained with Coomassie brilliant blue. Lanes: Std, molecular
weight standards; +Ni, sample from pKKGb cultures grown with nickel; -Ni,
sample from pKKGb cultures grown without nickel; the corresponding mutants
are indicated in each lane.

110

Std K20 D49 E88 072 080 S111 0+

    

kDa
107.0 _-
81.0 _-
48] --
33.8 —
20.7 — UreE
144'
H74 to
Std +Ni -Ni A c N UreE
UreE

 

Figure 3.10. Analysis of the UreE content in pull down samples. Western blotting
with anti-UreE polyclonal antibodies. Std, prestained molecular weight standards;
C+, purified sample of the UreE144* (15-residue truncated variant, (30)); +Ni,
sample from pKKGb cultures grown with nickel; -Ni, sample from pKKGb cultures
grown without nickel; UreE, purified sample of full-length UreE; the
corresponding mutants are indicated in each lane.

111

DISCUSSION

The studies described here greatly expand upon what is known about the
UreG urease accessory protein of K. aerogenes and provide new insights into
critical aspects of its function. In particular, this work sheds light on the lack of
necessity of dimerization (and corresponding disulfide bond formation) in the
protein, the roles of particular residues in binding metal ions or facilitating in vivo
activation of urease, and the ability of UreG to participate in a newly identified
activation complex.

The biotin-tagged form of UreG allows for easy purification of the wild-type
and mutant versions of the protein. The addition of the tag has no effect on the
tertiary structure of UreG, as shown by CD spectroscopy, but it does prevent
formation of a dimeric protein even in the presence of metal ions. Significantly,
the ability of UreGb to fully activate urease is a clear demonstration that the
dimer is not relevant for UreG function in vivo. The wild-type protein exhibits
identical gel filtration chromatography behavior regardless of the presence of
metal ions, but it slowly undergoes dimer formation concomitant with a decrease
in nickel ion binding capacity. This finding suggests that one of the two Cys
residues in UreG (located at positions 28 and 72) is involved in both dimerization
and metal binding. These results call into question the metal-binding results
obtained with dimeric, disulfide-containing UreG proteins previously
characterized from B. pasteurii (20) and M. tuberculosis (19).

Several additional differences exist between the K. aerogenes UreG and

the corresponding proteins from other organisms. UreG from K. aerogenes has

112

 

 

only 15% disordered structure as determined by CD spectroscopy, as opposed to
30% or 45% for UreG proteins from B. pasteurii (20) and M. tuberculosis (19),
suggesting that the K. aerogenes protein is more structured and perhaps better
suited to crystallographic studies. We found that Glu68, previously hypothesized
to be an important metal-binding residue at the comparable position in B.
pasteurii UreG (18), was nonessential for urease activation. In contrast, we used
mutagenesis approaches to demonstrate that several residues (Ly320, Asp49,
Cys72, His74, Asp80, and Ser111) in K. aerogenes UreG are critical for urease
activation, even though the variant proteins displayed little effect on their metal
ion binding properties. In the case of Lys20 and Asp49, roles in MgGTP binding
are likely on the basis of the HypB crystal structure. The finding that C72A,
H74A, and S111A UreGb variants bind nickel ions with parameters much like the
native protein suggests that the metal is simultaneously coordinated by many
residues, so that any single or certain double mutants exhibit no effects, or that
the metal binding sites are located elsewhere on the protein.

The newly created biotin-tagged form of UreG also was used to provide
evidence of novel protein-protein interactions in the cell. In particular, the pull-
down assays revealed the formation of a new urease activation complex with all
of the structural and accessory gene products including UreE. The use of this tag
will allow the purification and characterization of this key complex. Also of great
interest, the D80A UreGb variant possessed lower affinity for most proteins in the
complex, but retained the ability to bind UreE thus demonstrating a unique UreG-

UreE complex that can also be further studied. An interaction between UreG and

113

UreE was previously proposed on the basis of results from yeast two-hybrid
studies carried out with the H. pylori proteins (32, 33).

UreG and HypB share similarities in function (i.e., activation of a nickel-
containing enzyme), as revealed by the multiple sequence alignments. The K.
aerogenes UreG residues Cys72, His74, and Ser111 are likely to be equivalent
to M. jannaschii HypB residues Cys166, His167, and Cys198, and mutations to
the K. aerogenes residues abolished urease activity. Whereas these amino acids
coordinate a dinuclear zinc site in the M. jannaschii HypB crystal structure and
were identified as metal binding amino acids by mutagenesis studies of the E.
coli protein (23), our mutants show only a slightly smaller amount of nickel bound
per molecule (1.5 Ni/molecule compared to 2.0 Ni/molecule for UreGb). In
addition, the GTPase domain of HypB binds only one nickel atom per protein,
suggesting that at least one nickel binding site in UreG is found only in this

protein.

ACKNOWLEDGMENTS
I would like to thank Scott Mulrooney, Kimberly Anderson, and Rachel

Morr for their assistance.

114

REFERENCES

1. Hausinger, R. P., and Karplus, P. A. (2001) Urease, in Handbook of
Metalloprateins (Wieghardt, K., Huber, R., Poulos, T. L., and Messerschmidt, A.,
Eds), pp 867-879, John Wiley & Sons, Ltd., West Sussex, UK.

2. Jabri, E., Carr, M. B., Hausinger, R. P., and Karplus, P. A. (1995) The
crystal structure of urease from Klebsiella aerogenes, Science 268, 998-1004.

3. Benini, S., Rypniewski, W. R., Wilson, K. S., Miletti, S., Ciurli, S., and
Mangani, S. (1999) A new proposal for urease mechanism based on the crystal
structures of the native and inhibited enzyme from Bacillus pasteurii: why urea
hydrolysis costs two nickels, Structure 7, 205-216.

4. Ha, N.-C., Oh, S.-T., Sung, J. Y., Cha, K.-A., Lee, M. H., and Oh, B.-H.
(2001) Supramolecular assembly and acid resistance of Helicobacter pylori
urease, Nature Structure Biology 8, 505-509.

5. Sheridan, L., Wilmont, C. M., Cromie, K. D., van der Logt, P., and Phillips,
S. E. V. (2002) Crystallization and preliminary X-ray structure determination of
jack bean urease with a bound antibody fragment, Acta Crystallogr. D58, 374-
376.

6. Kim, J. K., Mulrooney, S. B., and Hausinger, R. P. (2005) Biosynthesis of
active Bacillus subtilis urease in the absence of known urease accessory
proteins, J. Bacteriol. 187, 71 50-71 54.

7. Quiroz, S., Kim, J. K., Mulrooney, S. B., and Hausinger, R. P. (2007)
Chaperones of nickel metabolism, in Metal Ions in Life Sciences (Sigel, A., Sigel,
H., and Sigel, R. K. O., Eds), pp 519-544, John Wiley & Sons, New York.

8. Hausinger, R. P., Colpas, G. J., and Soriano, A. (2001) Urease: a
paradigm for protein-assisted metallocenter assembly, ASM News 67, 78-84.

9. Lee, M. H., Mulrooney, S. B., and Hausinger, R. P. (1990) Purification,
characterization, and in vivo reconstitution of Klebsiella aerogenes urease
apoenzyme, J. Bacteriol. 172, 4427-4431.

10. Jabri, E., and Karplus, P. A. (1996) Structures of the Klebsiella aerogenes
urease apoprotein and two active-site mutants, Biochemistry 35, 10616-10626.

115

11. Park, l.-S., Carr, M. B., and Hausinger, R. P. (1994) In vitro activation of
urease apoprotein and role of UreD as a chaperone required for nickel
metallocenter assembly, Proc. Natl. Acad. Sci. USA 91, 3233-3237.

12. Moncrief, M. B. C., and Hausinger, R. P. (1996) Purification and activation
properties of UreD-UreF-urease apoprotein complexes, J. Bacterial. 178, 5417-
5421.

13. Moncrief, M. B. C., and Hausinger, R. P. (1997) Characterization of UreG,
identification of a UreD-UreF-UreG complex, and evidence suggesting that a
nucleotide-binding site in UreG is required for in vivo metallocenter assembly of
Klebsiella aerogenes urease, J. Bacteriol. 179, 4081-4086.

14. Soriano, A., Colpas, G. J., and Hausinger, R. P. (2000) UreE stimulation
of GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein
complex, Biochemistry 39, 12435-12440.

15. Soriano, A., and Hausinger, R. P. (1999) GTP-dependent activation of
urease apoprotein in complex with the UreD, UreF, and UreG accessory
proteins, Prac. Natl. Acad. Sci. USA 96, 11140-11144.

16. Kim, J. K., and Hausinger, R. P. (2006) The UreEF fusion protein provides
a soluble and functional form of the UreF urease accessory protein, J. Bacterial.
188, 8413-8420.

17. Salomone-Stagni, M., Zambelli, B., Musiani, F., and Ciurli, S. (2007) A
model-based proposal for the role of UreF as a GTPase-activating protein in the
urease active site biosynthesis, Proteins 68, 749-761.

18. Zambelli, B., Stola, M., Musiani, F., De Vriendt, K., Samyn, B., Devreese,
B., Van Beeumen, J., Dikiy, A., Bryant, D. A., and Ciurli, S. (2005) UreG, a
chaperone in the urease assembly process, is an intrinsically unstructured
GTPase that specifically binds Zn”, J. Biol. Chem. 280, 4684-4695.

19. Zambelli, B., Musiani, F., Savini, M., Tucker, P., and Ciurli, S. (2007)
Biochemical studies on Mycobacterium tuberculosis UreG and comparative
modeling reveal structural and functional conservation among the bacterial UreG
family, Biochemistry in press.

116

20. Neyroz, P., Zambelli, B., and Ciurli, S. (2006) lntrinsically disordered
structure of Bacillus pasteurii UreG as revealed by steady-state and time-
resolved fluorescence spectroscopy, Biochemistry 45, 8918-8930.

21. Gasper, R., Scrima, A., and Wittinghofer, A. (2006) Structural insights into
HypB, a GTP-binding protein that regulates metal binding, J. Biol. Chem. 281,
27492-27502.

22. Leach, M. R., and Zamble, D. B. (2007) Metallocenter assembly of the
hydrogenase enzymes, Curr. Opinion Chem. Biol. 11 , 159-165.

23. Leach, M. R., Sandal, 8., Sun, H., and Zamble, D. B. (2005) Metal binding
activity of the Escherichia coli hydrogenase maturation factor HypB, Biochemistry
44, 12229-12238.

24. Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999)
Identification of metal-binding residues in the Klebsiella aerogenes urease nickel
metallochaperone, UreE, Biochemistry 38, 4078-4088.

25. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly
of the head of bacteriophage T4, Nature (London) 227, 680-685.

26. Whitmore, L., and Wallace, B. A. (2004) DICHROWEB, an online server
for protein secondary structure analyses from circular dichroism spectroscopic
data, Nucl. Acids Res. 32, W668-673.

27. Weatherburn, M. W. (1967) Phenol-hypochlorite reaction for determination
of ammonia, Anal. Chem. 39, 971-974.

28. Maier, T., Drapal, N., Thanbichler, M., and Book, A. (1998) Strep-tag Il
affinity purification: an approach to study intermediates of metalloenzyme
biosynthesis, Anal Biochem 259, 68-73.

29. Leipe, D. D., Wolf, Y. l., Koonin, E. V., and Aravind, L. (2002)
Classification and evolution of P-l00p GTPases and related ATPases, J Mol Biol
317, 41-72.

117

30. Brayman, T. G., and Hausinger, R. P. (1996) Purification, characterization,
and functional analysis of a truncated Klebsiella aerogenes UreE urease
accessory protein lacking the histidine-rich carboxyl terminus, J. Bacterial. 178,
5410-5416.

31. Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y., and
Hausinger, R. P. (1992) Klebsiella aerogenes urease gene cluster: sequence of
ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG)
are involved in nickel metallocenter biosynthesis, J. Bacteriol. 174, 4324-4330.

32. Rain, J.-C., Selig, L., de Reuse, H., Battaglia, V., Reverdy, C., Simon, S.,
Lenzen, G., Petel, F., Wojcik, J., Schachter, V., Chemama, Y., Labigne, A., and
Legrain, P. (2001) The protein-protein interaction map of Helicobacter pylori,
Nature 409, 211-215.

33. Voland, P., Weeks, D. L., Marcus, E. A., Prinz, C., Sachs, G., and Scott,
D. (2003) Interactions among the seven Helicobacter pylori proteins encoded by
the urease gene cluster, Liver Physiol. 284, 696-G106.

34. Lu, J., Zheng, Y., Yamagishi, H., Odaka, M., Tsujimura, M., Maeda, M.,
and Endo, l. (2003) Motif CXCC in nitrile hydratase activator is critical for NHase
biogenesis in vivo, FEBS Lett 553, 391-396.

118

Chapter 4

Additional studies, conclusions and remaining questions

119

ADDITIONAL STUDIES

1. Maltose binding protein-UreF (MBP-UreF) fusion protein crystallization
attempts.

Previous efforts to isolate UreF for characterization demonstrated that this
protein is soluble only as a fusion protein with large tags such as thioredoxin or
Maltose binding protein (MBP)(1, 2). I decided to use the MBP-UreF fusion
protein for structural characterization through crystallization. The use of the MBP
to express and purify UreF has many advantages: it enhances solubility and
expression in the host; it allows purification with an affinity column giving high
yield and purification fold in a single step. Additionally, upon crystallization,
molecular replacement can be used to solve the structure, which'is a simpler and
less time-consuming method.

The use of fusion proteins also has some disadvantages such as impairment
of the protein function. The possibility of obtaining crystals is also reduced in the
case of the fusion protein. Multidomain proteins are usually less conductive to
farming well-ordered, diffracting crystals, presumably due to the conformational

heterogeneity allowed by the flexible linker region.

a. Expression and purification of MBP-UreF
Isolated colonies of E. coli DH5a containing the plasmid pMal-UreF (1)
were grown in 250 mL of Terrific broth supplemented with 100 ug/mL ampicillin at
37°C for 5 hours and then induced overnight with 10 or 100 mM isopropyl-B-D-

thiogalactopyranoside (IPTG). The cells were harvested by centrifugation and

120

resuspended in 20 mL of buffer PED (100 mM potassium phosphate pH 7.2, 1
mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT))
containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The suspension was
sonicated and centrifugated at 100,000 x g for 45 minutes at 4°C. The
supernatant was loaded onto an amylase column (Bio-Rad) previously
equilibrated in PED buffer. The fusion protein was eluted with PED buffer
containing 10 mM maltose. The collected fractions were analyzed by 12% or
15% SDS-PAGE stained with coomasie blue. Final protein quantification was
done with Bio-Rad Protein assay method using BSA as standard.

The fusion protein MBP-UreF was produced in higher amount when
cultures were induced with 10 mM IPTG (Figure 4.1, lane 1). More than 95% of
the protein was in the soluble fraction. After elution from the amylase resin, MBP-
UreF was more than 95% pure as judged by inspection of SDS-PAGE (Figure
4.2). A culture of 250 mL produced 7.5 mg of fusion protein. Protein UreF could
be separated from MBP by Factor Xa proteolysis but could not be recovered from
the following DEAE-Sepharose chorrnatography. This is in agreement with a

previous report (1).

121

MBP-UreF

 

Figure 4.1. SDS-PAGE of MBP-UreF expression on E. coli after induction. Lane
1, soluble fraction of cultures induced with 10 mM IPTG; lane 2, insoluble fraction
of cultures induced with 10 mM IPTG; lane 3, soluble fraction of cultures induced
with 100 mM IPTG; lane 4, insoluble fraction of cultures induced with 100 mM
IPTG.

kDa 1 2
158.0
116.0

66.4

55.6

42.7

36.5

26.6
14.3

I“ <— MBP-UreF

Figure 4.2. SDS-PAGE of purified MBP-UreF. Lane 1, molecular weight markers;
lane 2, purified MBP-UreF.
b. Crystallization of MBP-UreF.

Crystallization screening experiments were set up in Dr. Michael
Garavito’s lab using microbatch sitting drop vapor diffusion method. Each
experiment consisted of mixing 2 uL of MBP-UreF at either 15, 20 or 25 mglmL
with 2 uL of 138 crystallization screen solutions obtained from a combination of

commercially available crystallization kits. Protein drops were covered with 50/50

122

paraffin/silicon mix and stored at room temperature or 4°C for two months. The
status of each plate was checked every week.

After eight weeks, approximately 50% of the drops remained clear. No crystal
was found although protein precipitates were observed on the drops. The same

results were obtained with plates incubated at either room temperature or 4°C.

2. Crystallizations attempts for UreG

The accessory protein UreG was purified as described in chapter 3 to >90%
purity as determined by SDS-PAGE. A 2ul aliquot of a 10 mglml solution of the
protein in buffer Tris 100 mM, pH 8.0 containing 150 mM NaCl and 1 mM DTT
was mixed with 2pl of 168 crystallization solutions in Dr. Garavito's lab using a
microbatch sitting drop vapor diffusion method . Protein drops were covered with
50/50 paraffin/silicon mix and stored at room temperature for two weeks. The
status of the plates was checked every 5 days.

When the drops were set up, most of them presented precipitation, however
when the plates were checked after five days, crystals or promising precipitates
were observed under several condition detailed in table 4.1. No follow up was
possible for lack of time.

Table 4.1. Conditions that showed UreG crystals or promising precjaitantes.

 

 

 

 

 

 

 

 

 

Precipitant Buffer Salt/additive
1.5 M (NH4)ZSO4 0.1M Tris pH 8.5 12% glycerol

1.0 M (NH4)2PO4 0.1M citrate pH 5.5 200 mM NaCl
1.0 M (NH4)2PO4 0.1M lmidazole pH 8.0 200 mM NaCl
0.4 M (NH4)2PO4 None None

2.0 M (NH4)ZSO4 0.1M Tris pH 8.5 None

2.0 NaFormate 0.1 M Na acetate pH 4.6 None

1.6 M NaH2PO4/ 0.1 M Phosphate-citrate pH 4.2 None

0.4 M K2HPO4

 

 

 

 

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3. UreG homology model
Before the publication of the HypB crystal structure, I created a homology

model for UreG with the assistance of Dr. Michael Feig. The sequences of K.
aerogenes UreG was submitted to the Meta server Bioinfo (http:/lbioinfopl/Metal)
(3) for a primary structure analysis. The Meta server provides access to several
fold recognition and local structure prediction methods. Each method processes
the amino acid sequence separately with a specific algorithm. The fold
recognition servers search for structures that can be adopted by the submitted
amino acid sequence. The local structure prediction methods propose a
secondary structure for the protein. The results are collected and then translated
into uniform formats to evaluate them for structural similarity. When different
methods predict the same structure, the result is considered more reliable and a
high score is assigned. The Meta server also gathers primary structure search
results for sequence homologs. A more complete structure prediction for UreG
was done using the Multiscale Modeling Tools for Structural Biology (MMTSB)
tool set (4). The crystal structure of the signal sequence binding protein th from
Thennus aquaticus (Protein Data Bank code: 2th) was used as template. The
alignment of secondary structure elements, taken from the Bioinfo Meta server,
was used to generate a structure scaffold where side chains in the template
structure were replaced with the corresponding amino acids in K. aerogenes
UreG. Connecting loops and other missing fragments were then added with the
MMTSB Tool Set by sampling different conformations and selecting the most

favorable based on similarity and a force-field scoring function.

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The PDB-Blast search for sequence homologs of UreG gives high statistical
confidence values to several GTPases. The four proteins with the highest
similarity scores (all GTPases) were: the GTP-binding protein EngA from E. coli,
Era GTPase from E. coli, ina protein from E. coli, and the large 7 subunit of
initiation factor eif2 from Pyrococcus abyssi. None of the mentioned proteins
have more than 20% identity with UreG; therefore, none can be used to build a
homology model because this method requires at least a 30% sequence identity
to be reliable.

All three secondary structure prediction methods used in the Bioinfo Meta
server predict a protein with alpha helices and beta strands. The Meta server
evaluation of the structural models proposed by several fold recognition servers
gives a high score to structures based on ina protein from E. coli (mentioned
before within the high ranked proteins in the PDB-Blast search), the signal
recognition particle receptor from E. coli and the signal sequence binding protein
from Therrnus aquaticus (Ffl'l). Since the secondary structure prediction was in
better agreement with Ffl1, we decided to use this protein as the template for the
model.

The final model of the structure (Figure 4.3) shows a core of parallel beta
strands surrounded in both sides by alpha helices. The methodology used to
build the structural model of UreG cannot give atomic details of the structure, but ,
the overall topology is considered very reliable. In agreement with this affinnation,
when the model is compared to the structures in the Protein Data Bank (PDB)

using the Dali Server (http://wwweblacuk/dalil) only P-loop containing

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nucleotide binding proteins appear to be the most similar in structure with the

UreG model.

 

Figure 4.3. UreG model and HypB structure. The top panels show the UreG
model in light blue, HypB is shown in purple in the lower panels. Both structures
were turned in 180 degrees for the right side panels. Metal binding residues are
shown in green and GTP binding residues are shown in pink. The hypB structure
also shows the GTP analog in orange.

There are three highly conserved motifs within GTPases: the Walker A motif
or P-Ioop, the Walker B motif and the NDxK motif. The P-loop binds the
phosphate groups of the nucleotide. The Walker B motif (involved in Mg"2

binding) was modified in UreG as E)o<G instead of the more common DxxG

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sequence. The NKxD motif confers specificity to GTP by direct interaction with
the nucleotide reassuring that GTP is the natural substrate of UreG.

The conserved motifs between F111 and UreG are the GTPase-related P-loop,
Walker B and NKxD motifs. All were localized near from each other in the protein
model, in an adequate position to interact with GTP. Since there is no obvious
obstacle for GTP binding in the model, the proposed conformation is probably
adopted by UreG when is in the apoDFG complex and not isolated when is
unable to bind GTP (5).

The metal binding residues are not very well aligned as shown in figure 4.4.
However, Cys72 and His74 are in a flexible loop that could be easily rearranged.

Ser111 is not far from Cys127 position.

 

Figure 4.4. Partial superimposition of HypB crystal structure and UreG model.
UreG is colored light blue and HypB is in purple. The metal binding residues are
shown as sticks and the two spheres are the zinc ions found in the crystal.

127

CONCLUSIONS AND REMAINING QUESTIONS

The studies presented in this thesis resulted in numerous findings that
allow us to propose a more detailed model for urease metallocenter assembly.

In chapter 2, in silico flexibility analysis of the urease crystal structure by
our collaborators suggested that a flexible hinge in UreB may be involved in
urease activation by allowing the subunit to change its conformation and open up
the active site. This proposed conformational change agrees with an earlier
proposal from our lab that was based on results from chemical cross-linking
studies that showed two residues were in close contact, but only when facilitated
by the addition of UreF to the (UreABC-UreD)3 complex. My exchange of GIy11
and GIy18 for proline residues in UreB had mixed effects. In the case of the
G18P UreB variant within the context of the other urease proteins, I observed no
changes in urease activity. In contrast, the G11P UreB mutation caused a
significant reduction in urease activity, a decrease in nickel content, and the
recovery of a new activation complex (UreABC-UreDFGMUreEn from cell cultures.
I interpret these results as suggesting that even though the accessory proteins
can bind urease, the activation process is somehow hindered in this mutant. The
SAXS analyses carried out by our collaborator at Oak Ridge National
Laboratories provided data that positioned the UreD and UreF accessory
proteins at the vertices of the urease trimer, very close to each other and to UreB.
All together, these analyses are consistent with our initial hypothesis and
previous cross-linking data.

Chapter 3 describes an improved purification method for UreG that adds a
short amino acid sequence at the C-terminus of the protein which becomes
biotinylated in the cell. The addition of the tag did not modify the tertiary structure
of the protein nor did it interfere with urease activation. Based on sequence
alignments and comparison to the crystal structure of the related protein HypB, I
mutated selected conserved amino acids in UreG and determined the effects on
urease activation. The biotin tag also allowed me to test the interaction of these
mutant proteins with urease apoprotein and the other accessory proteins. Finally,

I measured the nickel-binding abilities of selected mutants. I found that the

128

majority of the mutations affecting UreG nearly abolished the ability of this protein

to facilitate urease activation, but all of the purified UreG mutants tested

(including double mutants) retained the ability to bind at least 1.6 nickel ions per

molecule (compared to 2 nickel ions per molecule for the non-mutated protein).

Therefore, these results suggest that these residues may not be the ligands used

for nickel binding, and the metal binding sites remain to be clearly identified. On

the other hand, I showed that mutations in the GTP binding amino acids are key
to urease activation. In one case (the D80A UreG mutant) I observed a lower
affinity for the complete activation complex, but continued interaction with UreE.
I’ve made substantial progress in characterizing several urease apoprotein
complexes and some properties of wild-type and mutant UreG species; however,
numerous questions remain to be answered about the urease activation process.

Here I propose some experiments that could help others to a better understand

the fascinating process of the urease activation, which may have relevance to the

activation of other metalloproteins:

a. The G11P mutant of UreB allows the isolation of a new activation complex
that contains all the structural subunits and accessory proteins. If this
complex can be characterized further (e.g., analysis by SAXS, chemical
modification studies, or even crystallization) it would give very valuable
information. Thus, I have created a potentially very useful construct for other
researchers to investigate.

b. The urease SAXS studies that I’ve pioneered can be extended in new
directions to obtain additional insights. A soluble form of UreF (a fusion of the
UreE and UreF proteins) is known to bind (UreABC-UreD)», to form the
(UreABC-UreD(E)F)3 complex. The extra domain provided by UreE could be
used to better localize UreF with the (UreABC-UreDF)3 complex. Of particular
interest might be the isolation of UreEF from cells grown in deuterated buffer,
formation of the larger complex with (UreABC-UreD)3 from normal buffer, and
analysis using neutron scattering methods. This approach has the potential to
more clearly define the position of UreEF in the large complex. Similarly,

129

other fusion proteins (such as maltose binding protein-UreD) could be use in
a similar approach.

. Two nickel ions are bound to each UreG molecule according to my
membrane centrifugation/PAR studies. The crystal structure of HypB hints at
the location of one set of metal ligands, but the mutation studies I performed
do not provide evidence to confirm that those targeted residues coordinate
the metal. More mutational, spectroscopic, and structural analyses are
required to clarify this point. For example, mutations could be constructed to
alter the second Cys in the protein, and several other potential nickel ion-
binding residues could be targeted. A type of spectroscopy known as X-ray
absorption spectroscopy can provide information about ligands to metal ions,
and this might be a particularly useful approach to identify whether the metal
is bound by imidazole ligands (the method cannot distinguish among most N
and O ligands, but the secondary scattering from the ring carbons allows one
to identify and quantify His ligands). My CD studies suggest that, unlike the
highly unstructured situations for other UreG proteins, the K. aerogenes UreG
might be amenable to crystallographic analysis. Indeed, I obtained preliminary
results suggesting that a particular crystallization buffer held promise (data
not shown). The structure of apoprotein or nickel ion-bound protein would be
valuable.

. The interaction between UreG and UreE now can be further characterized

thanks to the D80A variant of UreG. These two accessory proteins are likely
to be at the core of the activation process because they allow for the coupling
of nickel ion delivery to urease apoprotein and GTP hydrolysis. Crystallization
of these isolated proteins or co-crystallization of the UreG-UreE complex
could give better insight into the process of nickel introduction into urease. It
is possible that the inclusion of GTP could trigger conformational changes of
UreG within the UreG-UreE complex that could provide enhanced
understanding of the function of GTP in nickel insertion into the apoprotein.

. The D80A mutant can also be used to better understand the interaction

between UreG and the (UreABC-UreDF)3 activation complex, again making

130

use of several fusion proteins available in our laboratory (i.e. UreEF, MBP-
UreF, MBP-UreD) to determine if the interaction is with UreD, UreF or the
(UreABC-UreDF)3 complex.

f. The approach I used to target amino acids for site-directed mutagenesis of
UreG could be used to analyze UreD and UreF and possibly assess their
function. Despite of the low level of identity between UreD and UreF orthologs,
it is possible to identify some highly conserved residues that can be mutated.
It is important to note that no proteins of related sequence have yet been
crystallized for these two proteins and the insoluble nature of the proteins

prevents more structural analyses.

In conclusion, the information provided by my studies has improved our
understanding of urease metallocenter assembly. In addition, several mutants
and the new purification system for UreG could be used as tools for future
research of the system.

131

REFERENCES

1.

Kim, K. Y., Yang, C. H., and Lee, M. H. (1999) "Expression of the
recombinant Klebsiella aerogenes UreF protein as a MalE fusion" Arch
Pharm Res 22, 274-278.

Moncrief, M. B. C. "Unpublished results".

Ginalski, K., Elofsson, A., Fisher, D., and Rychlewski, L. (2003) "3D-Jury:
a simple approach to improve protein structure predictions" Bioinfonnatics
19, 1015-1018.

Feig, M., Karanicolas, J., and Brooks, C. L., Ill. (2004) "MMTSB tool set:
enhanced sampling and multiscale modeling methods for applications in
structural biology" J Molec Graphics Modeling 22, 377-395.

Moncrief, M. B. C., and Hausinger, R. P. (1997) "Characterization of UreG,
identification of a UreD-UreF-UreG complex, and evidence suggesting that
a nucleotide-binding site in UreG is required for in vivo metallocenter
assembly of Klebsiella aerogenes urease" J Bacteriol 1 79, 4081-4086.

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