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STRUCTURAL ASPECTS OF THROMBIN AND KRINGLE DOMAINS:
INHIBITION, LIGAND BINDING MODES AND THEIR BIOLOGICAL
RELEVANCE.

By

Igor Mochalkin

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemistry

1999

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ABSTRACT

STRUCTURAL ASPECTS OF THROMBIN AND KRINGLE DOMAINS:
INHIBITION, LIGAND BINDING MODES AND THEIR BIOLOGICAL
RELEVANCE.

By

Igor Mochalkin

The X-ray crystal structures of two thrombin active site inhibitors (SEL2711 and
SEL2770) have been determined and refined in the (9.0-2.1) A resolution range to final
R-values of 16.5% and 16.7%. Unlike most thrombin inhibitors, SEL2711 and SEL2770
bind in a retro-fashion, forming a parallel B-strand with residues Ser214-Gly216 of
thrombin. The thrombin bound crystal structures of SEL2711 and SEL2770 were used to
correlate the binding constants and contributions of the different thrombin subsites to the
binding mode. Since the Selectide inhibitors are about 104 times more specific for Factor
Xa, modeling of the inhibitors to the latter suggests that the selectivity can be a
consequence of interactions in the 83-84 binding subsites of Factor Xa that are different
in thrombin and Factor VIIa.

The X-ray crystal structures of two B-strand templated thrombin active site
inhibitors (MOL376 and MOL592) have been determined and refined in the (9.0-2.3) A
and (9.0-2.0) A resolution ranges to final R-values of 15.3% and 17.4%. Unlike

SEL2711 and SEL2770, MOL376 and MOL592 bind to thrombin in a substrate-like

fashion, making an anti-parallel B-strand with residues Ser214-Gly216 of the enzyme.

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The network of interactions between the inhibitors and thrombin at the SI subsite was
used to correlated the trend in the binding constants and provide important insight for the
future design of therapeutic antithrombotic agents. Probing the operating sites of
thrombin further with urea showed that three urea molecules bind to the active site region
of the enzyme and two are located next to the heparin binding exosite II. The overall
pattern of the interaction at the specificity site is similar to that found in crystal structures
of thrombin-PPACK and many other thrombin-inhibitor complexes in which an Arg
residue occupies the P1 position.

The X-ray crystal structure of the recombinant kringle (K) 5 domain of human
plasminogen has been determined by molecular replacement methods using K1 as a
starting model and refined in the (8.0-1.7) A resolution range to a final R-value of 16.6%.
The presence of Leu71 at the ligand binding site (LigBS) leads to a substantial decrease
of the binding affinity of K5 for s-aminocaproic acid (EACA), with respect to K1 and
K4. Based on the crystal structure, the Li gBS was rationally remodeled and a K5[L71R]
mutant possesses an affinity for w-amino acids that is similar to K1 and K4.

Three X-ray crystal structures of the recombinant KIV-lO variants of human
apolipoprotien (a) have been determined and refined in the (9.0-1.8) and (9.0-2.1) A
resolution ranges to final R-values of (16.6-18.2)%. The crystal structures indicate that:
(1) both KIV-lO/Thr66 and KIV-lO/Met66 can bind EACA in the embedded way, (2)
Arg72 in the KIV-lO/W72R mutant occupies the LigBS mimicking ligand and (3) Arg35

shifts upon EACA binding to assist Arg7l at the cationic center.

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ACKNOWLEDGMENTS

In a project of this magnitude, many individuals make significant contributions to
its success. Foremost among those whom I would like to acknowledge for help is Prof.
Alexander Tulinsky, my graduate adviser. Prof. Tulinsky not only suggested the ideas
behind most of the studies but he also provided a constant flow of recommendations and
suggestions and financial support so that most of the work could be done under a research
assistantship. I also owe a special debt of gratitude to the members of his team: Dr.
Robert St. Charles, Dr. Jorge L. Rios-Steiner and Dr. Raman Krishnan for their guidance,
encouragement, generosity and advice in technical, academic and career matters
throughout my graduate career.

There are many other people to whom I wish to express my thanks for their help.
I wish to express my appreciation to Dr. Irimpan I. Mathews and Dr. Kaillathe
Padmanabhan for their help in my earliest project: retro-binding inhibition of thrombin by
SEL2711 and SEL2770. I wish to express my thanks to Dr. Raman Krishnan and Dr.
Raghuvir K. Ami for their contributions (MOL354, MOL1245 and MOL356) to the study
of the non-electrophilic Molecumetics inhibitors as they related to my research project
with MOL376 and MOL592. Furthermore, the projects involving plasminogen kringle 5
and apolipoprotein (a) KIV-lO greatly benefited from the expertise in protein
crystallization of Dr. Beisong Cheng, who crystallized these kringle modules. I must also
mention my appreciation to Dr. Peggy Vanderhoff-Hanaver who introduced me to the
crystallization techniques and helped me to build a strong foundation to become
knowledgeable in this field. I would like to thank Prof. Francis J. Castellino and his

research group from the University of Notre Dame, Prof. Angelo Scanu and Dr. Olga

iv

 

coord
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and 1
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WI
my 1

Walt

Klezovitch from the University of Chicago and Dr. Brian R. Boettcher of Novartis
Pharmaceuticals Corp., (Summit, NJ.) for providing protein samples of plasminogen
kringle 5 (F.J.C) and apolipoprotein (a) KIV-lO (AS and OK.) and unpublished atomic
coordinates and observed structure factors of unbounded KIV-lO/Thr66/EACA (B.R.B).
I would like to thank the Selectide Corp. and Molecumetics Ltd. for providing the SEL
and MOL inhibitors and Dr. Herman Schreuder of Hoescht Marion Roussel (Frankfurt,
Germany) and Dr. Hiroshi Nakanishi of Molecumetics Ltd. (Bellevue, WA.) for a
number of helpful discussions. Thanks are also due to Dr. F. Fratemali of the National
Institute for Medical Research (London, England) for providing the coordinates of the
NMR structure of Factor Xa complexed with peptide A and B. The writing of most of
my published papers was supported by the James L. Dye Endowed Fellowship and the
Walter and Margaret Yates Memorial Scholarship.

I gratefully acknowledge the influence of Prof. Gerald Davis (Department of
Medical Technology, MSU) and Prof. Houria Hassouna (Clinical Center, MSU) who
expanded my knowledge in hematology and hemostasis and maintained an interest in my
progress. I also wish to express my thanks to my best friends and my loved ones: Dianna
Johnson, René St.Louis and Alexander and Millicent Popov for their support.

And finally, I wish express my most profound thanks to my parents and my sister
who since my earliest recollections have always encouraged my curiosity, quest for
fundamental knowledge and my attempts to learn. They have backed me financially when
necessary, but more importantly, they have backed me with intangibles that I can never

repay. It is to my parents that I dedicate this work.

ACKOXV
TABLE C
LIST OF 1
LIST OF '
ABBRB
CHAPTI
l. HERIO'

I.‘.

TABLE OF CONTENTS

ACKONWLEDGEMENT .................................................................. iv
TABLE OF CONTENTS .................................................................. vi
LIST OF FIGURES ......................................................................... viii
LIST OF TABLES ........................................................................... xiv
ABBREVIATIONS AND SYMBOLS ................................................... xvii

CHAPTER 1: BLOOD PROTEINS AND COAGULATION

I. HEMOSTASIS ............................................................................. 1
1.1 . Endothelium ..................................................................... l
1.2. Platelet ........................................................................... 3
1.3. The Coagulation Cascade ...................................................... 8

II. FIBRINOLYSIS .......................................................................... 12

III. MODULAR NATURE OF BLOOD PROTEINS .............................. 13

CHAPTER 2: RETRO-BINDING INHIBITION OF THROMBIN

I. INTRODUCTION ........................................................................ 22
1.1. Thrombin Binding Sites ....................................................... 22
1.2. Thrombin Active Site Inhibitors and Their Binding Modes .............. 24
1.3. Selectide Corp. Inhibitors ..................................................... 25
II. EXPERIMENTAL ....................................................................... 28
III. RESULTS ................................................................................ 33
IV. DISCUSSION .......................................................................... 39

vi

 

CHAPTEI

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IV.

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IV,

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II. E
III. ;

CHAPTER 3: SELECTIVE NON-ELECTROPHILIC INHIBITORS OF

THROMBIN
I. MOLECUMETICS INHIBITORS ............................................. 52
II. EXPERIMENTAL .............................................................. 55
III. RESULTS ........................................................................ 56
IV. DISCUSSION .................................................................. 65

CHAPTER 4: PROBING THE ACTIVE SITE OF THROMBIN FURTHER WITH

UREA
I. INTRODUCTION ............................................................... 73
II. EXPERIMENTAL ............................................................... 76
III. RESULTS AND DISCUSSION .............................................. 84

CHAPTER 5: STRUCTURE-FUNCION DETERMINANTS OF RECOMBINANT

KRINGLE 5 OF HUMAN PLASMINOGEN

I. INTRODUCTION ................................................................ 94
II. EXPERIMENTAL ................................................................ 97
III. RESULTS ........................................................................ 103
IV. DISCUSSION ................ . ................................................... 108

CHAPTER 6: RECOMBINANT KRINGLE IV-10 MODULES OF HUMAN
APOLIPOPROTEIN (a): STRUCTURES, LIGAND BINDING

MODES AND BIOLOGICAL RELEVANCE

I. INTRODUCTION .................................................................. l 15
II. EXPERIMENTAL ................................................................ 1 19
III. RESULTS ......................................................................... 124

vii

 

 

IV.
GLOSSAII

BIBLIOGI

CHAPTER

Figure I-l.

Figure 1-2.

Figure 1.3.

IV. DISCUSSION ..................................................................... 133

GLOSSARY .................................................................................... 140

BIBLIOGRAPHY ............................................................................. 144

LIST OF FIGURES

CHAPTER 1: BLOOD PROTEINS AND COAGULATION

Figure l-l.

Figure 1-2.

Figure 1-3.

Figure 1-4.

Figure 1-5.

Functions of endothelium. Anticoagulant factors and their modes of
action in the coagulation cascade are represented in bold black.
Coagulation factors secreted by the endothelial cells are in thin line text.
Double dot denotes binary complex.

Megakaryocyte cell development from the level of the stem cells, through
BFU-MK, CFU-MK, and then into the morphologically recognizable
platelet precursor cell in stage I-IV.

Progress of platelet plug formation in hemostasis. A — platelet activation
and secretory function; B — platelet adhesion; C — platelet aggregation; D —
platelet clot formation.

Intrinsic and extrinsic pathways of the blood coagulation cascade.

The prothrombinase complex. Prothrombin is composed of fragment 1 V
(F1), fragment 2 (F2) and prethrombin 2 (Pre-2). FVa = Va; FXa = Xa;
calcium ions are filled circles. FVa is embedded into the phospholipid

layer.

viii

 

Figure I-6.

Figure l-7.

Figure 1-8.

Figure 1-9.

CHAPTER
Figure 2-1.

Figure 2.1

“We 2.3

Figure 1-6.

Figure l-7.

Figure 1-8.

Figure 1-9.

CHAPTER 2:
Figure 2-1.

Figure 2-2.

Figure 2-3.

Figure 2-4.

Structural motifs of various blood proteins.

Molecule of human LDL. Apo(a) consists of 14 to 41 kringle 4-like units,
referred to as KIV, distributed into 10 classes (KIV-l...KIV-10). KIV-9
links apo(a) and apo B-100. KIV-lO is followed by one copy of KV and a
protease domain.

Kringle domain. Primary structure of human apo(a) KIV-lO.

The photograph of normal (A) and 70% occluded (B) coronary arteries. In
latter, white and black streaks represent cholesterol depositions and

hemorrhage into the atherosclerotic plug, respectively.

RETRO-BINDING INHIBITION OF THROMBIN

Active site retro-binding inhibitors of thrombin.

Stereoview of the final (2F o-Fc) electron density of the (A) SEL2711 and
(B) SEL2770 inhibitors in the thrombin complexes. Contoured at the 10
level; SEL2711 is shown in bold black. SEL2770 is shown in bold black.
Thrombin residues are in thin gray and numbered.

Stereoview of superimposed Selectide inhibitors in the active site of
thrombin. SEL2711 and SEL2770 are shown in bold black and gray,
respectively. Thrombin residues of the SEL2711 complex are in thin gray
and numbered.

Stereoview of superimposed SEL2711 and BMS-183507 bound to

thrombin. SEL2711 and BMS-183507 are shown in bold black and gray,

ix

 

Figure 2-1

Figure 2.

CHAPT

Flfigure 3.

Figure 3-.

Figure 2-5.

Figure 2-6.

respectively. Thrombin residues of the SEL2711 complex are in thin gray
and numbered.

Stereoview of SEL2711 and SEL2770 superimposed on the active site of
FXa in a retro-binding mode. SEL2711 and SEL2770 are shown in bold
black and gray, respectively. FXa residues are in thin gray and numbered.
Since the pyridine of SEL2770 collides with Phel74 of FXa, a
conformational change must occur in one or the other, most likely the
former.

Stereoview of SEL2711 and SEL2770 superimposed on the active site of
FVIIa in a retro-binding mode. SEL2711 and SEL2770 are shown in bold
black and gray, respectively. F VIIa residues are in thin gray and

numbered.

CHAPTER 3: SELECTIVE NON-ELECTROPHILIC INHIBITORS OF

Figure 3-1.

Figure 3-2.

THROMBIN

Selective non-electrophilic Molecumetics active site inhibitors of
thrombin. Electrophilic inhibitor D-Phe-Pro-Arg chloromethyl ketone
(PPACK) is shown for comparison. Numbering is defined on MOL376;
K, is shown for both thrombin and trypsin (upper and lower number,
respectively); ratio of Ki '3 is on the right indicates a degree of selectivity.
The Ramachandran plot of the final structure of the thrombin- (A)
MOL376 and (B) MOL592 complexes. Glycine residues are shown as

triangles; non-glycine residues as squared boxes.

 

 

Figure 3-3.

Figure 3-4.

Figure 3-5.

Figure 3-6.

FIEUIe 4.2

Figure 3-3.

Figure 3-4.

Figure 3-5.

Figure 3-6.

Stereoview of the final (2Fo-Fc) electron density of the (A) MOL376 and
(B) MOLS92 inhibitors in the thrombin complexes. Contoured at the 10'
level; MOL376 and MOL592 are in bold black. Thrombin residues are in
thin gray and numbered.

Stereoview of superimposed MOL354/376/ 1245 bound to thrombin.
MOL354 is in bold black; MOL376 is broken black line; MOL1245 is in
bold gray. Thrombin residues of the MOL1245 complex are in thin gray
and numbered.

Stereoview of superposed MOL356 and MOL592 bound to thrombin.
MOL356 is in bold black; MOL592 is in bold blue. Thrombin residues of
the MOL356 complex are in thin gray and numbered.

Schematic diagram of hydrogen bonded salt bridges of Molecumetics
inhibitors with Asp189 of the 81 specificity site of thrombin.
MOL354/ 1245 shown together in one schematic, MOL1245 in

parentheses.

CHAPTER 4: PROBING THE ACTIVE SITE OF THROMBIN FURTHER WITH

Figure 4-1.

Figure 4-2.

UREA

Urea binding sites of thrombin. Urel- Ure3 are in the active site region of
the enzyme. Urea4 and UreS are located adjacent to heparin binding
exosite II. Serl95 of the catalytic triad is numbered.

Stereoview of the final (2Fo-Fc) electron density of Urel-Ure3 and their

water environment. Contoured at the 10 level.

xi

Figure 4-3

CHAPTEI

Figure 5-1.

Figure 5-2.

Figure 5-3.

 

Figure 5-4.

Figure 5.:

V0

 

Figure 4-3. Schematic diagram of hydrogen bonds (A) formed by Urel and Ure2 in

the active site region of the thrombin-himgen complex.

CHAPTER 5: STRUCTURE-FUNCION DETERMINANTS OF RECOMBINANT
KRINGLE 5 OF HUMAN PLASMINOGEN

Figure 5-1. Primary structure of plasminogen K5.

Figure 5-2. Space filling and ribbon representations of plasminogen K5 dimer viewed
approximately down the local 2-fold axis in the crystal structure.

Figure 5-3. Superposition of the LigBS of K5 molecules 1 and 2 of the dimer. A
residue of each segment is labeled. Molecule 1 is shown in gray. Molecule
2 is colored in black.

Figure 5-4. The Ramachandran plot of the final structure of the K5 dimer. Lys48
residues of molecule 1 (Lysl48) and molecule 2 (Ly3248) are in a
generously allowed region. Glycine residues are shown as triangles; non-
glycine residues are shown as squared boxes.

Figure 5-5. Stereoview of specific residues of K5 potentially present in the LigBS of
molecule 1 of the K5 dimer. The regions of the kringle represented are
His33-Phe36, AspSS-Asp57, Trp62-Tyr64, Leu71-Tyr74 shown in gray.
A residue of each segment is labeled. Water molecules common to the
LigBS of both kringles of the dimer are 1, 4, 5, 7, and 8; those in molecule
1 only are 2, 3, and 6. 1:0,..540, 2=Ow525, 3=Ow4l9, 4=Ow565,
5=Ow589, 6=Ow524, 7=Ow526, 8=Ow527 of the PDB coordinate file (PDB

code: SHPG).

xii

Figure 5-6.

CHAPTER 1

Figure 6-1.

 

Figure 6.2,

I
k

Figure 5-6.

Superposition of the LigBS of K1 and K5 of plasminogen. K5 is in gray;
K1 is shown in black. K1 is inhibited with EACA in the binding pocket.

All residues are numbered according to KS.

CHAPTER 6: RECOMBINANT KRINGLE IV-10 MODULES OF HUMAN

Figure 6-1.

Figure 6-2.

Figure 6-3.

APOLIPOPROTEIN (a): STRUCTURES, LIGAND BINDING
MODES AND BIOLOGICAL RELEVANCE

Superposition of EACA of different lysine binding kringles. KIV-
lO/Thr66/EACA side chains of the LigBS are in gray. Embedded binding
modes are labeled: EACA of K1 (1); EACA of K4 (2); EACA of KIV-
10/Thr66/EACA (3). EACA of KIV-lO/Thr66/EACA (Sandoz) displays
the unbound binding mode. Note different conformations of Arg36 in
KIV-lO/Thr66/EACA and in KIV- l O/Met66.

Stereoview of the final (2Fo-Fc) electron density of EACA of KIV-
lOfI'hr66/EACA displaying the embedded binding mode. Contoured at the

10 level. Two conformations of Arg32 are shown.

Superposition of KIV-lO/W72R (dark gray) on KIV-lO/Met66/EACA

(light gray) showing Arg72 approximating EACA binding. Note different

conformation of Arg36 in the two structures.

xiii

 

CH
Tat

Tat

CH
Tab

Tab.

Iabl

Tabl

CH1

Table

Tahie

LIST OF TABLES

CHAPTER 1: BLOOD PROTEINS AND COAGULATION

Table 1-1.

Table 1-2.

Morphologic criteria for megakaryocytes and platelets.

Conserved structural modules in different blood proteins.

CHAPTER 2: RETRO-BINDING INHIBITION OF THROMBIN

Table 2-1.

Table 2-2.

Table 2-3.

Table 2-4.

Specificity of Selectide inhibitors (Ki(uM)).

Crystal data and intensity data collection statistics of SEL2711 and
SEL2770.

Refinement summary of deviations from ideality of thrombin molecules in
the SEL2711 and SEL2770 complexes.

Intermolecular and intramolecular interactions in the active site of the
thrombin complexed with SEL2711 and SEL2770 inhibitors.

Numbering and nomenclature as used in text.

CHAPTER 3: SELECTIVE NON-ELECTROPHILIC INHIBITORS OF

Table 3-1.

Table 3-2.

THROMBIN
Intensity data collection of Molecumetics inhibitors MOL376 and

MOL592 bound to thrombin.
Refinement summary of deviations from ideality of thrombin

molecules in the MOL376 and NOL592 complexes.

xiv

Table 3-3.

Individual refinement statistics of MOL3 76 and MOL592 inhibitors.

CHAPTER 4: PROBING THE ACTIVE SITE OF THROMBIN FURTHER WITH

Table 4-1.

Table 4-2.

Table 4-3.

UREA

Crystal data and intensity data collection statistics of the native and
urea-treated thrombin-hirugen.

Rmcrge-value as a function of the (A) structure factors; (B) resolution;
(C) F/o(F) of the native and urea-treated crystals of thrombin-himgen.
Refinement summary of deviations from ideality and final refinement

parameters of the thrombin-hirugen-urea complex.

CHAPTER 5: STRUCTURE-FUNCION DETERMINANTS OF RECOMBINANT

Table 5-1.

Table 5-2.

Table 5-3.

Table 5-4.

Table 5-5.

KRINGLE 5 OF HUMAN PLASMINOGEN

Kd (uM) values of ligand binding to various kringle modules.

Primary structures of plasminogen Kl-KS and apo(a) KIV-lO aligned
in respect of K1 to maximize conservation. The number of residues (1-
80) standardized to plasminogen K5. Deletion sites at positions 34 and
59 are indicated with —. Sequence conservation is boxed: identical
residues are in the black boxes, homologous residues are in the gray
boxes.

Primary structures of the plasminogen K5 modules from the various
species.

Crystal data and intensity data collection statistics of plasminogen K5.

Refinement summary of deviations from ideality and individual

XV

Ial

CH

Fab.

Table

Table

Table .
Table I

Table (

Table 5-6.

refinement statistics of plasminogen K5.

Hydrogen bonds (A) in the LigBS region of K5.

CHAPTER 6: RECOMBINANT KRINGLE IV-10 MODULES OF HUMAN

Table 6-1.

Table 6—2.

Table 6-3.

Table 6-4.

Table 6-5.

Table 6-6.

APOLIPOPROTEIN (a): STRUCTURES, LIGAND BINDING

MODES AND BIOLOGICAL RELEVANCE

Primary structures of various kringles aligned in respect of apo(a)
KIV-10 to maximize conservation. The number of residues (1-80)
standardized to plasminogen K5. Deletion sites at positions 34, 42A,
47A, 59 and 70A are indicated with —. Sequence conservation is
boxed: identical residues are in the black boxes, homologous residues
are in the gray boxes.

Conditions used to crystallized the various KIV-10 kringles.

Crystal data and intensity data collection statistics of different KIV-10
crystals.

Summary of final restrained least-square statistics of KIV-10 modules.
Probable conserved hydrogen bonds (A) in LigBS of various KIV-10.
Probable hydrogen bonds (A) in LigBS of KIV dependent on ligand

binding and the Trp72Arg mutation.

xvi

ABBREVIATIONS AND SYMBOLS
7-AHpA, 7-aminoheptanoic acid; 6-AHx, 6-aminohexane; t-AMCHA, trans-4-
aminomethylcyclohexane-l-carboxylic acid; S-APn, S-aminopentane; S-APnA, 5-
aminopentanoic acid; apo(a), apoB- l 00. apolipoprotein B-lOO; apolipoprotein(a);
Argatroban,(2R,4R)-4-methyl-l ma-[O-methyl-l ,2,3,4-tetrahydro-8-
quinolinyl)sulfonyl]-L-arginyl]-2-piperidine carboxylic acid; ATIII, antithrombin
III;
B, temperature factor; BF U-MK, burst-forming-unit megakaryocyte;
CF U-MK, colony-forrning-unit megakaryocyte; Chg, L-cyclohexylglycyl; CSF,
colony stimulatory factors;
d, resolution;
EACA, e-aminocaproic acid; EGF, epidermal growth factor; EGR, glutamyl-
glycyl-arginyl chloromethylketone;
F, structure factors; F Va, factor Va; F VII, factor VII; F VIIa, factor VIIa; FIX,
factor IX; FX, factor X; FXa, factor Xa; FXII, factor XII; FXIa, factor XIa; FXI,
factor XI; FXIII, factor XIII; FXIIIa, factor XIIIa; FVIII-FIX-FX, X-ase complex;
Gla, y-carboxyglutamic acid;
hirugen, sulfated Tyr63-N-acetyl-hirudin 53-64;
IFN, interferon; IL, interleukins:
k, scale constant; K1 to K5, the kringle l to kringle 5 regions (residues Cy884-
CysS4l) of human plasminogen; K5[L71 R] recombinant Leu7lArg kringle 5
mutant; KIV and KV, kringle 4 and kringle 5 of apo(a); KIV-l, KIV-2,...KIV-10,

KIV modules, type 1-10; KL. C-kit ligand;

xvii

I??.<<S-'

LDL, low density lipoprotein; LigBS, ligand binding site; Lp(a), lipoprotein(a);
MO, macrophages; Meg-POT, megakaryocytes potentiator; MSF, maturation
stimulatory factors;

NAPAP, Nu-(2-naphthyl-sulfonyl-glycyl)-D-para-amidinophenylalanyl-
piperidine; NCS, non-crystallographic symmetry; NK, natural killer cell;
les(2Me), L-(N,N-dimethyl)lysine; NO, nitrogen (11) oxide;

Pal3(Me), 3-(methyl pyridinium)alanyl; pAph, L-4-amidino-phenylalanyl; PEG,
polyethylene glycol; PGIZ, prostacyclin; PGPMC, platelet glycoprotein-bearing
mononuclear cell; PT, prothrombin; ; PPAC K, D-phenylalanyl-prolyl-arginyl
chloromethylketone; PT-F 1 . prothrombin fragment 1; PT-F2, prothrombin
fragment 2;

rmsA, root-mean-square deviation;

SPB, sodium phosphate buffer;

T, T-lymphocytes; TF, tissue factor; t-PA, tissue-type plasminogen activator;
TPO, thrombopoetin;

u-PA, urokinase plasminogen activator;

vWF, van Willebrand factor;

X-ase complex, FVII-FIX;

Q, occupancy parameter.

xviii

CHAPTER 1

BLOOD PROTEINS AND COAGULATION

I. HEMOSTASIS
Under normal physiological conditions, blood of higher organisms is in a fluid
state. Upon tissue damage or vascular injury, a hemostatic response is primed to arrest
hemOrrhage, seal off the site of damage and begin a process Of tissue repair. The delicate
and highly dynamic balance between blood clotting and dissolution is regulated by four
major processes: endothelial maintenance, platelet functions, the coagulation cascade

(hemostasis) and fibrinolysis.

1.1. ENDOTHELIUM

Endothelium is an interfacing single layer of smooth thin cells between the blood
vessel lumen and the circulating blood. This unique location demands that endothelial
cells particrpate in both hemostasis and vascular permeability (Colman et al., 1994). On
the one hand, endothelial cells possess an adhesive texture on their outer or ablurninal
surface, which faces the subendothelial tissues. On the other hand, the luminal surface,
which is exposed to the bloodstream, is highly non-thrombogenic. Thus, endothelium
serves as a shield for circulating platelets against exposure to subendothelial activation
factors and a barrier for separation of blood proteins from the tissue thromboplastin or
other surface contact activators that, otherwise, would provoke the coagulation cascade.

Endothelium performs both anticoagulant and procoagulant activities due to its

synthetic and secretory functions (Figure 1-1). Among the anticoagulant factors

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synthesized by endothelial cells are prostacyclin (P612), tissue-type plasminogen activator
(t-PA) and urokinase plasminogen activator (u-PA), nitrogen (H) oxide (NO),
thrombomodulin and heparin. As a procoagulant, endothelium also synthesizes and
secretes from its abluminal surface collagen, thromboplastin, fibronectin, von Willebrand
factor (vWF), and elastin (Stemerman et al., 1984; Colman et al., 1994).

Endothelium injury could occur in various forms by either direct or indirect
induction and range from mechanical (sloughing), physical (freezing, heat or radiation),
chemical (carbon monoxide, hypercholesterolemia) to immunological and viral. Under
normal physiological conditions, maximum sheer wall stress in the arterial tree is about
200 dynes/cm3. Sheer stress exceeding 400 dynes/cm3 can tear endothelium from the
arterial wall exposing the thrombogenic subendothelial matrix to the bloodstream.
Sloughing of the non-thrombogenic endothelial layer induced by either mechanical,
atherogenic injury, hypertension or thrombotic disorders deprive the vessels of numerous

intrinsic anticoagulant protective mechanisms and initiate platelet plug formation.

1.2 PLATELETS
Platelets are derived from their hematopoietic precursor cells, megakaryocytes
that have been established to comprise approximately 0.02% — 0.05% of bone marrow
nucleated cell population (Harker, 1968). Through a series of poorly understood events,
lineage-indifferent stem cells give rise to more differentiated progenitor cells, the burst-
forming-unit megakaryocyte (BFU-MK) and the colony-forrning-unit magakaryocyte
(CPU-MK), that are committed to development within a given lineage (Figure 1-2).

Platelet glycoprotein-bearing mononuclear cells (PGPMC) are transitional between the

 

’ INF Alpha Granule Proteins

Stem Cell /0
0—01 0

O
BFU- MK 0 DD
V. o o O .1. . o 00
CFU- MK 0 CO

 

 

 

 

 

 

 

 

 

 

 

 

PGPMC
Megakaryocyte Morphologically Recognizable Platelets
progenitor cells Megakaryocytes
IL- la TPO
lL-l 1 MSF
KL Meg-POT
CSF IL-6

 

 

 

 

 

 

Stimulatory Proteins

5 l
® cg) Kidney & Liver

Ancillary Marrow Cells
(Lymphocytes, Monocytes, Natural Killer
Cells, Stromal Cells, Endothelial Cells)

 

 

Figure 1-2: Megakaryocyte cell development from the level of the stem cells,
through BFU-MK, CFU-MK and then into the morphologically recognizable

platelet precursor cell in stages I-IV.

progenitor cells of the first developmental compartment

and morphologically

recognizable cells of the second developmental compartment where megakaryoblasts,

promegakaryocytes and megakaryocytes are undergoing the terminal phases of maturation

before releasing platelets into the blood (Table l- 1 ).

Table 1-1: Morphologic criteria for megakaryocytes and platelets.

 

Characteristics

Megakaryocytes

Platelets (Thrombocytes)

 

Cell Size (diameter)

35-160 um

2-4um

 

Nuclear-Cytoplasrnic Ratio

1:1—1:3

 

Nuclear Shape

Lobulated (2 or more lobes)

 

Nucleoli

None

 

 

 

 

Some known modulators of human megakaryocytopoiesis are T-lymphocytes (T),
macrophages (MO), natural killer cells (NK), interferon (IFN), c-kit ligand (KL),
megakaryocytes potentiator (Meg-POT), maturation stimulatory factors (MSF), colony
stimulatory factors (CSF), thrombopoietin (TPO), endothelial cells and various
interleukins (IL) (Figure 1-2).

The ability of platelets to form a platelet plug is critical to the maintenance of
normal hemostasis (Born & Hardisty, 1972; Colman et al., 1994). Platelet procoagulation
activity includes three major steps: activation, adhesion and aggregation (Figure l-3/A-
C). When vascular injury occurs and endothelium is damaged, thrombogenic

subendothelial tissues such as collagen and basement membrane are exposed to the

 

 

 

  

 

BLOOD COAGULATION CASCADE

FIBluN

 

 

 

 

 

Figure 1-3: Progress of platelet plug formation in hemostasis.

A-platelet activation and secretory function; B-platet adhesion;

C-platelet aggregation; D -p1atelet clot formation.

6

 

bloodstream. Platelets circulating in the blood come into a contact with the exposed
subendothelial factors and become activated. Activated platelets: (l) adhere to the
surface by the binding of vWF to platelet glycoprotein Ib/D( (2) expose and secrete
certain membrane-bound glycoproteins and (3) undergo a change in shape. The new
incoming platelets then aggregate at the site of the injury (Figure 1—3/C) through a
molecule of fibrinogen linked to glycoproteins IIb/IIIa of two adjacent platelets. Initial
'platelet plug formation at the site of the endothelial damage is referred to as primary
hemostasis.

To form an effective plug, the circulating platelets must be present in adequate
numbers (100-400 x 109/L) and have normal functions. Decreased platelet counts are
referred to as thrombocytopenia and is the most common platelet disorder of insufficient
platelet production and excessive removal of platelets from circulation. Failure of
sufficient platelet production can result from ( 1) decreased thrombopoietin, (2) decreased
number of megakaryocytes or (3) ineffective thrombopoiesis. The following factors
could contribute to removal of platelets from the circulation: (1) platelet destruction, (2)
excessive platelet consumption (disseminated intravascular coagulation), (3) loss
(hemorrhage) or (4) dilution with platelet-poor blood during massive blood transfusion.
Increased platelet counts are classified as thrombocythemia.

While thrombocytopenia is associated with decreased numbers the circulating
platelets, thrombocytopathy refers to abnormal platelet functions in adhesion and
aggregation. These defects could be classified as inherited or acquired. The inherited
defects include Benard-Soulier Syndrome, pseudo-von Willebrand disease, Glanzmann’s

thrombasthenia, release disorders and storage pool deficiencies. The acquired defects

occur when the platelet functions are affected by drugs, diet or disease. In
thrombocytopathy the platelet count is usually normal or slightly increased, but

occasionally both conditions are present simultaneously.

1.3. THE COAGULATION CASCADE

To strengthen the platelet plug formed at the site of vascular injury, a series of
enzymatic reactions initially triggered by tissue factor (TF) result in a cross-linked fibrin
clot formation (Figure 1-3/D) (MacFarlane et al., 1972). This fibrin network stabilizes
the platelet plug against dissolution and is crucial in the coagulation process.

Two similar schemes of the blood coagulation cascade were independently
proposed approximately 35 years ago (Davie & Ratnoff, 1964; MacFarlane, 1964). Each
proposal suggested two principal mechanisms to stop the loss of blood: these pathways
are referred to as extrinsic and intrinsic (Figure 1-4). Present evidence indicates that the
extrinsic pathway plays an important role in the initiation of coagulation, while the
intrinsic pathway is responsible for growth and maintenance of the fibrin clot.

In general, the coagulation cascade is a series of stepwise reactions that utilize
protein molecules circulating in the blood in order to seal an area of endothelial damage
and to stop blood loss (Davie et al., 1991). The extrinsic pathway requires TF, a
membrane glycoprotein with a molecular weight of approximately 44,000 Da that is
located in the tissue adventita and comes into contact with blood after a vascular injury.
The extracellular domain of TF spans from residue Serl to Glu219, the trans-membrane
region of 23 amino acids extends from Ile220 to Leu242 and the cytoplasmic portion is

21 residues in length (His243 to Ser263) located at the carboxyl end of the protein.

Blood Coagulation Pathways

Intrinsic Pathway

Prekallikrein

    

Extrinsic Pathway

fissue
factor

 

 

 

 

prothrombin (ll)
fibrinogen (I) /\

THROMBUS J l

DISSOLVED
TH FIOMBUS

Figure 1-4: Intrinsic and extrinsic pathways of the blood coagulation cascade.

   
    
 

 

‘1

  

; plasmin

     

plasminogen

 

Residue CysZ45 is acylated by either palmic or steric acid. When a vascular injury occurs
and TF is released at the site of the endothelial damage, Factor VII (FVII) forms a one-to-
one complex with TF in the presence of calcium ions (Ca2+). This reaction facilitates the
conversion of FVH, by minor proteolysis, to an active serine protease, Factor VHa
(FVIIa). Subsequently, the FVIIa-TF complex (Banner et al., 1996; Zhang et al., 1999)
triggers the activation of Factor X (FX, Stuart factor) to Factor Xa (FXa) by the cleavage
of a single Arg52-Ile53 peptide bond.

In contrast to the extrinsic pathway that is initiated by vascular injury followed by
a release of TF, the intrinsic pathway is initiated by the components contained entirely
within the vascular system (Figure 1-4). The zymogen Factor XII (FXII, Hageman factor)
is the protein that triggers the series of stepwise reactions of the intrinsic pathway. It
binds to negatively charged surfaces such as kaolin, dextran sulfate and sulfatides and
initiates the sequence of reactions resulting in the activation of Factor IX (FIX) by Factor
XIa (FXIa). Thereafter, two pathways are merged at the site of the activation of FX
(Figure 1-4).

FXa (Mr. ~ 45,000 Da) is a vitamin K-dependent protein that is generated through
limited proteolysis by either the FVIIa-TF complex of the extrinsic pathway (Osterud &
Rapaport, 1977; Silverberg et al., 1977) or the X-ase complex (FVIIIa-FIXa) of the
intrinsic pathway (Stern et al., 1985). Among all of the obligatory feedback mechanisms
of blood coagulation, FXa activates prothrombin to thrombin through a so-called
prothrombinase complex (Mann et al., 1990) (Figure 1-5) and can also activate the WH-

TF complex. In the prothrombinase complex, FXa has been shown to be bound to Factor

10

 

Figure 1-5: The prothrombinase complex. Prothrombin is composed of fragment 1

(F1), fragment 2 (F2) and prethrombin 2 (pre-FZ). Factor Va=Va; Factor Xa=Xa;

calcium ions are filled circles. F Va is embedded into the phospholipid layer.

Va (FVa) and to the phospholipid surface via its y—carboxyglutarnic acid (Gla) containing
region. FXa also interacts with a molecule of prothrombin (PT), which is also bound to
the phospholipid layer via the Gla-domain of fragment 1 (PT F 1) and to FVa via the
fragment 2 region (PT F2 or PT K2).

In the common part of the coagulation cascade, thrombin, generated by through
the prothrombinase complex (Figure 1—4) cleaves fibrinogen to form fibrin that leads to
the fibrin network of the clot (Figure il-3/D and 1-4). Factor XIIIa (FXIIIa), generated
from its Factor XIII (FXIII) precursor by thrombin in the presence of calcium ions
(Lorand & Konishi, 1964; Naski et al., 1991), cross-links fibrin monomers by forming £-
(y—glutamyl)lysine bonds between two adjacent molecules (Folk & Finlayson, 1977).
These covalent cross-links result in the formation of the strong fibrin network that

stabilizes a pre-formed platelet plug.

11. FIBRINOLYSIS

The phenomenon of clot formation and dissolution is closely balanced and
regulated by the hemostatic and fibrinolytic systems. Clot dissolution is necessary for
tissue repair and the maintenance of normal blood flow to vital organs. The fibrinolytic
system includes: ( 1) plasma proteins such as the zymogen plasminogen and
proteolytically active plasrnin, (2) several plasminogen activators and (3) various plasmin
inhibitors (Bachmann, 1994).

The essential step in fibrinolysis is the activation of plasminogen to plasmin
(Figure 1-4). Plasrninogen consists of the N-terrninal peptide, an array of five different

kringle units (Kl-K5) followed by a protease domain that contains a catalytic triad of a

12

serine protease (Sottrup-Jensen et al., 1978). Two plasminogen activators, t-PA and u-
PA, cleave the Arg560-Va1561 bond of the zymogen to generate the active serine protease
plasmin. Plasmin then degrades the insoluble matrix of the fibrin clot into soluble
degradation products.

Physiologically, the fibrinolytic system is designed as a natural response to
thrombosis. When thrombotic complications occur, therapeutic use of plasminogen
activators becomes important. Due to the fast clearance and low effectiveness of
externally administrated t-PA, significant quantities of the activator are usually required
in practice. However, high dosage of t-PA could lead to hemorrhage, especially in older
people with a history of hypertension. From this standpoint, plasminogen activators and
inhibitors that can alter delivery mechanisms of plasmin(ogen) to the fibrin clot become

important.

III. MODULAR NATURE OF BLOOD PROTEINS

Because of the high sequence and structural homology among various serine
proteases participating in the blood coagulation cascade and fibrinolysis, it is likely that
proteases originated from a simple trypsin—like ancestor. During evolution, a varying
number of modules was inserted between the N-terminal and C-terrninal ends giving rise
to proteins with highly specialized functions (Figures 1-6 & 1-7, Table 1-2). The
structural modules of some blood serine proteases are: (1) Gla domain (2) the finger
domain (fibronectin type I and II), (3) the epidermal growth factor (EGF), (4) the apple

domain (5) the kringle module and (6) protease or catalytic domain.

13

”BMW—

FIX amm—

FX 900mm—

FVII amm—
Protein c momm—

MEAWM—

t-PA 5W»—
u-PA M659,—
Plasrninogen #3993599,—

 

El Signal peptide A Fibronectin (type I)

«m- Propeptide & Fibronectin (type H)
m GLA domain >4 Zymogen activation region
’6? EGF domain — Catalytic domain

E Kringle domain

Figure 1-6: Structural motifs of various blood proteins.

14

Phospholipids

Unest. cholesterol
LDL . 0 O~g
. . Q I . I 'f"
t ' 4); . {apo 8100
.e- ’
o.
*‘ \
Kringies 'i I Protease domain
IV
IV
IV

 

Human apo(a)

Figure 1-7: Molecule of human LDL. Apo(a) consists of 14 to 41 kringle 4-like
units, referred to as KIV, distributed into 10 classes (KIV-1, KIV-10). KIV-9
links apo(a) and apo B-100. KIV-10 is followed by one copy of KV and a protease

domain.

15

Table 1-2: Conserved structural modules in different blood proteins.

 

Structural Module Blood Proteins

 

 

Gla Domain PT, FVII, FD(, FX, proteins C, S and Z
Finger Domain Fibronectin, FXII
EGF FV H, FD(, FX, FXII, proteins C, S and Z, u-PA, t-PA, fibronectin
Apple Domain Prekallikrein and FXI .
Kringle Domain Apo(a); plasminogen, PT, hepatocyte growth factor, t-PA, u-PA,

vampire bat plasminogen activator, angiostatin

 

 

 

(l) The Gla domain is an autonomous structural and functional module of
approximately 40 residues found in various blood proteins (Figure 1-6; Table 1-2). The
module contains eight to ten Gla residues produced in post-translational modification of
glutarnic acid by a vitamin K-dependent carboxylase. In the case of the blood proteins,
these Gla residues are responsible for binding of proteases to platelets or phospholipid
membrane in a calcium (ID dependent manner (Sunnerhagen et al., 1995). It has been
shown that in the apo-structure of PT-Fl, most of the Gla domain structure is disordered
(Park & Tulinsky, 1986; Seshadn' et al., 1991). However, when the protein is crystallized
in the presence of Ca2+ (Soriano-Garcia et al., 1992) or strontium (II) (Sr2+) ions (Seshadri
et al., 1994), it exhibits a well folded Gla domain structure. Moreover, based on the
fluorescence and circular dichroism (Deerfield et al., 1987), there are two conformational

transitions involving two different metal binding sites of the Gla domain: (1) higher

16

affinity non-cation specific cooperative sites and (2) lower affinity Ca2+ and Sr2+ ion
binding specific sites. However, the mechanism by which these ions promote protein
binding to the membrane has yet to be fully elucidated in detail.

(2) The finger domains are homologous to fibronectin type I and type H domains
that bind cell surfaces and other molecules including collagen, fibrin, heparin, DNA, and
actin (Petersen et al., 1983).

(3) A sequence of about thirty to forty amino-acid residues found in the sequence
of epidermal growth factor has been shown (Blomquist et al., 1984; Doolittle et al., 1984;
Barker et al., 1986; Appella et al., 1988; Davis, 1990) to be present, in more or less
conservative form, in a large number of other proteins including those participating in the
coagulation cascade and fibrinolysis (FVII, FIX, FX, FXII, proteins C, S, and Z, u-PA and
t-PA) (Table 1-2). The functional significance of EGF domains in these apparently
unrelated proteins is not yet clear. A common feature, however, is that these repeats are
found in the extracellular domains of membrane-bound proteins or in proteins known to
be secreted.

(4) The apple domain, a 90 arnino-acid modular unit, has been found only in
prekallikrein and Factor XI (FXD which are two related plasma serine proteases activated
by FXIIa (Figure 1-4). The two share the same domain topology: an N-terminal region
with a quadruple repeat of the apple domains and a C-terrninal catalytic domain. The
apple domain contains six conserved cysteines linked together in a pattern 1-6, 2-5 and 3-
4 (McMullen et al., l99la,b).

(5) Kringles are autonomous structural and functional modular units that consist

of a characteristic triple loop structure of approximately 80 amino acids cross-linked by

17

three disulfide bridges in the pattern 1-6, 2-4 and 3-5 (Figure 1-8). Multiple copies of
kringle units exist in non-catalytic regions of a variety of blood proteins (Table 1-2). In a
striking example there are 14 to 41 kringle copies in apolipoprotein(a) (apo(a)) of
lipoprotein(a) (Lp(a)) (Figure 1-7). Plasrninogen possesses an array of five highly
homologous kringle units (Kl-K5) (Figure 1-6) (Sottrup-Jensen et al., 1978). A single
copy of a kringle exists in u-PA, vampire bat plasminogen activator and FXII. Kringles
are also found as pairs in PT and t-PA and as a quartet in hepatocyte growth factor
(Nakamura et al., 1989) and angiostatin (Lijnen et al., 1998).

Kringle domains are also involved in extracellular proteolytic processes such as
angiogenesis, artherosclerosis, tumor-growth and metastasis (Cao et al., 1996). In
angiogenesis, the formation of new blood vessels supplying nutrients to new cells occurs
as a result of the growth of capillaries by a vascular sprouting from pre-existing vessels.
Direct experimental studies suggest that tumor growth and metastasis are angiogenesis-
dependent processes. An abnormal growth of new blood vessels can result in progression
of the tumor, stimulated by angiogenic factors such as fibrinoblast growth and vascular
endothelial growth factors that synergistically promote tumor growth. On the other hand,
the process can be highly inhibited by negative angiogenesis regulators such as
angiostatin, an endogenous angiogenesis inhibitor of 38,000 Da that consists of the first
four kringle units (Kl-K4) of plasminogen.

Kringle modules are also found in proteins involved in development of
atherosclerosis, another disease threatening the human population in this century.
Atherosclerosis, the most common cause of coronary heart disease, myocardial infarction

and cerebrovascular disease, is characterized by cholesterol and lipid deposition on the

18

 

Figure 1-8: Kringle domain. Primary structure of human apo(a) KIV-10

l9

innermost layer of the walls of large and medium sized arteries and results in a vascular
obstruction and insufficient blood supply to organs (Figure 1-9). In human subjects, a
high incidence of cardiovascular disease has been correlated with high plasma levels of
Lp(a) (Dahlen et al., 1978; Kostner et al., 1981; Murai et al., 1986; Dahlen et al., 1986).
Lp(a) is a special class of low density lipoproteins (LDL) having as a protein moiety
apo(a) covalently linked to apoBlOO by a disulfide bond (Figure 1-7) (Brunner et al.,
1993; Koschinsky et al., 1993). Kringles are the building blocks of apo(a) the primary
sequence of which shares a remarkable degree of homology with those of plasminogen
(McLean et al., 1987; Eaton et al., 1987). Apo(a) lacks the first three kringles of
plasminogen and contains from 14 to 41 kringle 4-like repeats, referred to as KIV,
distributed into 10 classes designated 1 to 10, exhibiting 75% to 94% sequence homology
with plasminogen K4. KIV-9 contains an unpaired cysteine that links apo(a) to one of the
unpaired cysteines in the C-terrninal domain of apoBlOO. KIV-10 is followed by one
copy of KV and a protease domain, which have 91% and 94% sequence homology with

K5 and the catalytic domain of plasminogen, respectively (McLean et al., 1987).

20

 

(A)

 

Figure 1-9: The photograph of normal (A) and 70% occluded (B) coronary
arteries. In latter, white and black streaks represent cholesterol depositions

and hemorrhage into the atherosclerotic plug, respectively.

21

CHAPTER 2:

RETRO-BINDING INHIBITION OF THROMBIN

I. INTRODUCTION

Based on the protein hierarchy in the coagulation cascade, thrombin (Mr. ~36,000
Da) is a multifunctional serine protease that plays an important role. in both blood
coagulation and fibrinolysis (Figure 1-4). As a prominent coagulant, thrombin cleaves
fibrinogen, which leads to fibrin, and activates platelets, factors V, VII, VIII and XIII
(FV, FVII, FVIII and FXIII) (Colman et al., 1994). Bound to thrombomodulin, thrombin
promotes anti-coagulation by activating protein C that converts factor Va (FV a) and
factor VIIIa (FVIIIa) to inactive forms in the presence of cofactor protein S (Figure 1-1)

(Esmon, 1993).

1.1. THROMBIN BINDING SITES

Thrombin owes its multifunctional role to four distinctive operating sites: (1) the
active site, (2) the fibrinogen recognition site (exosite D, (3) the heparin binding site
(exosite II) and (4) a sodium (1) (Na+) ion binding site (Tulinsky, 1996). Like other
members of the trypsin-like family, thrombin possesses a catalytic triad of Hi557-Asp102-
Serl95 (chymotrypsinogen numbering, Bode et al., 1992) at the active site surrounded by
the Ser214-Cy5220 structural segment that forms a loop consisting of two almost
orthogonal arrays. The equatorial array of Ser214-Gly216 is critical in its association

with the main chain of substrates or peptidic inhibitors by forming an anti-parallel

22

(substrate binding) (Bode et al., 1989; Mathews et al., 1994) or parallel (retro-binding) [3-
sheet (Tabernero et al., 1995; Mochalkin & Tulinsky 1999). Both retro- and substrate-
like binding active-site inhibitors of thrombin usually possess two or three hydrogen
bonds via Ser214-O, Gly2l6-N and Gly216-O. The axial array of Glu2l7-Cy3220
restricts a specificity pocket that contains Aspl89 at its bottom, a key residue of thrombin
in the recognition event. An insertion loop at position 60, which partially occludes the
active site at the top, is responsible for some additional restricted active site specificity of
thrombin compared to that of trypsin or FXa (Locht et al., 1997).

Thrombin fibrinogen binding exosite I is responsible for the association of the
natural substrate fibrinogen (Blomback, 1967) and the potent natural inhibitor hirudin
(Rydel et al., 1991). The hirudin molecule simultaneously binds to the exosite I and the
active site of thrombin. On the other hand, hirugen, a useful fragment of hirudin (sulfated
Tyr63-N-acetyl-hirudin 53-64), binds solely to exosite I and thus prevents autocleavage of
thrombin with the active site unoccupied. Thrombin heparin binding exosite H binds
heparin, a prominent antithrombotic agent that causes a 50,000-fold increase in the rate of
inhibition of thrombin by antithrombin HI (ATIII) (Olson & Bjork, 1992). An allosteric
change from a kinetically slow to a fast form as a result of binding Na+ at a sodium
binding site of thrombin affects the energetics of the active site of the enzyme (Wells &
Di Cera, 1992). While fibrinogen binds to the fast form with high affinity (Mathur et al.,
1993) and is cleaved with higher specificity, the slow form activates the anticoagulant
protein C more specifically (Dang et al., 1995). Two Na+ binding sites have been

identified and described crystallographically with the aid of diffusion-exchange of the

23

Na+ ion by a rubidium (I) (Rb+) ion (Zhang & Tulinsky, 1997), one intramolecular, the

other intermolecular.

1.2. THROMBIN ACTIVE SITE INHIBITORS AND THEIR BINDING MODES

Since the determination of the crystal structure of thrombin (Bode et al., 1989), an
immerse array of various thrombin inhibitors has appeared (Balasubramanian, 1995).
Selective inhibition of thrombin has been the principal target of antithrombotic drug
design ventures aimed at essential replacing the clinical use of heparin, which is neither
highly optimal, convenient nor controllable. There are numerous serine proteases
unrelated to the coagulation cascade that play other important physiological roles in life
processes. Such enzymes could be accidental targets of less discriminating thrombin
inhibitors and could lead to unwanted side effects.

All peptidic thrombin active site inhibitors can be divided into two principal
classes according to their binding orientations with respect to residues Ser214-Gly216 of
thrombin (antiparallel or parallel B-strand). The antiparallel group, also operative with
factors VHa, D(a and Xa (FVHa, FIXa and FXa), consists of a variety of inhibitors that
mimic natural substrate fibrinogen at the P1-P3 positions, the archetype for thrombin
being D-phenylalanyI-prolyl-arginyl chloromethylketone (PPACK) (Bode et al., 1992)
and glutamyl-glycyl-arginyl chloromethylketone (EGR) for FXa. There are additionally a
large number of other small molecule peptide inhibitors of thrombin (Balasubramanian,
1995). This group includes Argatroban ((2R,4R)-4—methyl-l[Na-[(3-methyl-1,2,3,4-
tetrahydro-8-quinolinyl)sulfonyl]-L-arginyl]—2-piperidine carboxylic acid) and NAPAP

(Na-(Z-naphthyl-sulfonyl-glycyl)-D-para-amidinophenylalanyl-piperidine) (Brandstetter

24

et al., 1992) that form an antiparallel B-strand even though their binding mode is different
at the Sl’-S3 subsites (Mathews & Tulinsky, 1995). The second class of thrombin
inhibitors consists of hirudin-like active site binding inhibitors (Rydel et al., 1991) that
show a retro-binding mode of association by forming a parallel B-strand with Ser214-
Gly2l6. This class inhibitors includes the synthetic peptide HMS-183507 (Tabemero et
al., 1995), the natural product Nazumamide A (Nienaber et al., 1996) (Figure 2-1) and a

RPPGF breakdown fragment of bradykinin (unpublished results of this laboratory).

1.3. SELECTIDE CORP. INHIBITORS

A class of tri- to octa-peptide competitive inhibitors of FXa has recently been
identified from the screening of a large combinatorial library (Ostrem et al., 1998). The
consensus sequence is Tyr-Ile-Arg-X, where X is a hydrophobic amino acid and
isoleucine is replacable with leucine. The minimal sequence is the first three residues and
may be replaced by homologous non-natural amino acids. Some of these have been
shown to inhibit hydrolysis of chromogenic substrates of FXa but bind to the enzyme
differently from substrate such that Serl95 of the catalytic triad does not attack the
carbonyl group at the P1 position (Ostrem et al., 1998). Selectide Corp. (a subsidary of
Hoechst Marion Roussel) inhibitors SEL2711 and SEL2770 (Figure 2-1) are members of
this class of combinatorially designed and potent active site inhibitors of FXa that are also
highly selective against thrombin and other blood proteases (Table 2-1), exhibiting dose-

dependent efficacy following both intravenous and oral administration in a rat

25

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2.258 wmztow 0 mm .ESB med .wx 40 .o\\ so
a \ «:0 mo. \«0 mw. any
0 v34 «0 .¢O\
NW6 an” ES
“2‘ IIFz

N .I
326332 :z/_\zz 8mm: SE

:2

«22% :z

26

arteriovenous shunt thrombosis model (Seligmann et al., 1995). A reverse binding mode
similar to N-terrninal hirudin active site binding to thrombin was suggested to explain the
selectivity of this class of inhibitors (Ostrem et al., 1998). However, from an NMR study
of two of the inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH; and Ac-(4-amino-Phe)-(cyclohexyl-
Gly)-Arg-NH2 (peptides A and B, respectively), bound to the active site of FXa
(Fratemali et al., 1998), the authors state that "the inhibitors assume a compact, very well
defined conformation, embedded into the substrate binding 'site not in the same way as a
substrate", which was also not a retro-binding mode but with arginine inserted in the 31

specificity site.

Table 2-1: Specificity of Selectide inhibitors (Ki(uM))

 

 

 

 

Proteins SEL271 1 SEL2770
FXa 0.003 0.002
Thrombin 40 8
Protein C 10 12
Plasmin 130 60
FVHa/T F >200 >200
Trypsin 1 12 NA

 

27

 

Diffusion of the SEL2711 and SEL2770 inhibitors into crystals of FXa proved
unsuccessful, probably because, in tetragonal crystals, the active site is blocked by a
substrate-like intermolecular interaction (Padmanabhan et al., 1993). On the other hand,
co-crystallization attempts with the inhibitors also did not produce crystals. This led us to
soak the inhibitors into thrombin-hirugen crystals in an effort to possibly establish and
corroborate the different binding mode in thrombin and then model it as it might occur in

FXa.

II. EXPERIMENTAL

Crystallization: An approximately 10-fold molar excess of hirugen was added to a
frozen sample of human Ot-thrombin solution (1.0 mg/mL in 0.75 M NaCl) at 4° C to
form a 1:1 thrombin-hirugen complex and prevent autocleavage of the enzyme. The
solution of the complex was concentrated to about 3.5 mg/mL using a Centricon 10
concentrator with a molecular weight cutoff of 10 kDa in a refrigerated centrifuge at 4° C.
The thrombin-hirugen complex was crystallized using the hanging drop crystallization
method in a 4 uL drop, which consisted of 2 11L of the protein sample and 2 ILL of a well
solution. The well solution contained 28% polyethylene glycol (PEG) 8000 in 0.1 M
sodium phosphate buffer (SPB), pH 7.3. A repetitive macroseeding technique was
applied to enlarge the crystals. X-ray diffraction quality crystals with dimensions of 0.25
x 0.20 x 0.20 mm were transferred into a protein-free storing drop containing 32 % PEG
8000 in 0.1 M SPB, pH 7.3. The SEL2711 and SEL2770 inhibitors were soaked into
thrombin-hirugen crystals separately. A l uL aliquot of a 20 mM solution containing the

inhibitor was added to the storing drop and 1 uL of the resulting solution was removed

28

from the drop in intervals of 10-12 hours for 5 days until the final concentration of
inhibitor in the drop was about 15 mM.

Intensity Data Collection: X-ray diffraction data of the ternary SEL2711 and
SEL2770 thrombin-hirugen complexes were collected with an R-AXIS H imaging plate
detector. The radiation generated from a Rigaku RU200 rotating anode operating at 5 kW
power with the fine focus filament (0.3 mm x 3.0 mm) was monochromated (CuKa) and
intensified by focusing with Molecular Structure Corp. - Yale mirrors. The crystal to
detector distance was 10.0 cm and the detector-swing angle was zero degrees. Both
crystals scattered X-rays to 2.1 A resolution although the diffraction pattern of the
SEL2770 complex was considerably weaker (Table 2-2). Autoindexing and processing of
the measured intensity data were carried out with the Rigaku R-AXIS H software package
(Higashi, 1990). The intensity data collection statistics are summarized in Table 2-2.

Structure Determination: The crystal structure of the SEL2711 complex was
determined using thrombin coordinates of the isostructural binary thrombin-hirugen
complex (PDB code: 1HAH) (Vijayalakshmi et al., 1994). The initial coordinates were
optimized by rigid-body refinement using the X-PLOR program package (Brunger, 1992)
to an R-value of 30.4% (7.0-2.5) A followed by 3 cycles of coordinate and overall
temperature factor (B) refinement with a starting temperature factor B = 30.0 A2 using the
program PROFFT (R-value =27.4%) (Hendrickson, 1985; Finzel, 1987). After 6 cycles

of individual B refinement, the R-value converged at 21.6%.

29

Table 2-2: Crystal data and intensity data collection statistics of SEL2711 and SEL2770.

 

 

 

SEL271 l SEL2770
Space group C221 C28
Cell constants (A):
a 71.05 71.66
b 71.82 72.00
c 73.45 73.45
[3 (deg) 101.2 101.2
Resolution (A) 2.1 2. 1
Observations (I/O'(I)>1.0) 28,464 24,962
I/o(I) (outermost range) (2.3-2. 1) A 3.2 2.3
Independent reflections (I/O'(I)>1 .0) 17,688 13,507
Redundancy 1 .6 l .8
Completeness (%) 78 60
outermost range (%) 52 37
R-merge (%) 6.9 4.9
outermost range (%) 14.8 14.3

 

a - Four ternary complexes per unit cell, one per asymmetric unit.

30

The crystal structure of the SEL2770 complex was solved with the coordinates of
thrombin of the refined thrombin-SEL2711 structure as a starting structure. After 3
cycles of overall and 6 cycles of individual B refinement, the R-value was 20.0% (7.0-
2.5) A. Both complexes at this stage showed electron density corresponding to hirugen
and the active site inhibitors (which were omitted from calculations) in (2Fo-Fc) electron
and (Fo-Fc) difference density maps. Thereafter, the data were expanded to (9.0 — 2.5) A
and then 2.1 A resolution and the Na+ ions were located and solvent water molecules
were progressively found and added by examining difference density maps. The final
structure of thrombin complexed with SEL2711 contains 131 molecules of water with
occupancies (n) > 0.5, R=16.5% (9.0-2.1) A and <B>=26.8 A2. The final SEL2770
structure contains 104 water molecules with O > 0.5 refined to R=16.7% (9.0—2. 1) A and
<B>=34.8 A2. Both complexes also have two Na+ ions, one intramolecular and one
intermolecular essentially identical to those initially described (Zhang & Tulinsky, 1997).
The final refinement statistics of the complexes are presented in Table 2-3. The
coordinates of the ternary complexes have been deposited in the Brookhaven Protein Data

Bank (SEL2711, access PDB code: 7KME; SEL2770, access PDB code: 8KME).

31

Table 2-3: Refinement summary of deviations from ideality of thrombin molecules in

the SEL2711 and SEL2770 complexes.

 

 

Restraints Target SEL271 1 SEL2770
Distances (A):
Bond distance 0.020 0.011 0.014
Angle distance 0.030 0.037 0.036
Planar 1,4 distance 0.050 0.047 0.046
Planes 0.040 0.034 0.030
Chiral volume (A3) 0.15 0.17 0.17

Non-bonded contacts (A)

Single-torsion 0.60 0.22 0.24
Multiple torsion 0.60 0.25 0.28
Possible H-bond 0.60 0.25 0.28

Isotropic thermal factors (A2)

Main-chain bond 1.0 1.0 0.9
Main-chain angle 1.5 1.7 1.5
Side-chain bond 2.0 2.5 2.1
Side-chain angle 3.0 3.6 3.0

 

32

III. RESULTS

Thrombin: The electron density is well defined for most of the residues of
thrombin in both complexes except for the termini of the A chain (residues ThrlH-
AlalB, lDe4K-Arg15) and the autolysis loop (residues Trp148-Lysl49E), which are
typically disordered in other isomorphous thrombin structures. The two complexes show
no large conformational differences from each other as well as from other thrombin
structures. The root-mean-square deviation (rmsA) of CA positions between the two
structures is 0.25 A for 253 atom pairs. Comparing the thrombin structures of the ternary
complexes with that of the binary thrombin-hirugen complex (PDB code: 1HAH) gives
rmsA's of 0.21 A and 0.30 A for the SEL2711 and SEL2770 complexes, respectively.
The Ramachandran plots (Laskowsky et al., 1993) of the SEL2711 and SEL2770
complexes show that 196 (81.7 %) and 189 (78.8 %) residues of the total 240 non-glycine
and non-proline amino acids of thrombin are in most favorable regions while 44 (18.3 %)
and 51 (21.2 %) residues occupy additionally allowed areas.

Two Na” ion-binding sites, one (intramolecular) responsible for the slow to fast
kinetic transition (Wells & DiCera, 1992), and the other intermolecular, have been
identified in both structures. The Na+ ions adopt distorted octahedral arrangements
coordinated by solvent water molecules and the carbonyl oxygen atoms of Arg221A,
Ly3224 (intramolecular ion) and from Lysl69, Thr172 of one molecule and Phe204A of a
symmetry related molecule for the intermolecular site and are the same as those described
previously (Zhang & Tulinsky, 1997). The occupancies and the temperature factors of

the Na“ ions of the SEL2711 and SEL2770 complexes are: (2:1.00, B: 23.1 A2 and

33

12:0.79, B=27.6 A2 (intramolecular) and (2:084, B=22.9 A2 and 0:0.82, B=27.2 A2
(intermolecular), respectively.

Selectide Inhibitors: Both SEL2711 and SEL2770 bind to thrombin in a retro-
fashion forming a parallel B-strand with residues Ser2l4-Gly216. The electron density of
SEL2711 is well defined except that there is none corresponding to the C-terrninal
leucine and proline residues (Figure 2-2/A). The (2Fo-Fc) electron density map of
SEL2770 has a break at the amide between the L-(N,N-dimethyl)lySine (les(2Me)3)
group (nomenclature and numbering used here is the same as that in Ostrem et al., 1998)
and the leucine residue and although the density corresponding to C-terrninal dipeptide is
weaker than the remainder of the inhibitor, it is sufficient to trace the conformations of
the leucine and proline (Figure 2-2/B).

As in most crystal structures of thrombin complexed with active site inhibitors
containing an arginine-like residue, the specificity site of the enzyme is occupied by
the arginine-like L-4-arnidino-phenylalanyl (pAphl) residue in both complexes and
utilizes doubly hydrogen bonded ionic interactions between the arnidino group and
side chain of Asp189 (Figure 2-3, Table 2-4). In addition to this hydrogen bonded salt
bridge, one of the nitrogen atoms of the arnidino group also makes contact with
Gly219-O and Ala190-O while the other nitrogen forms a hydrogen bond to a water
molecule that bridges to Phe227-O (Table 2-4). The Glul92 residue of thrombin
adopts an extended conformation into the solvent region, instead of the more common
bent one covering the SI binding site (Vijayalakshmi et al., 1994), and provides space

for the L—cyclohexylglycyl (Chg2) residues of SEL2711 and SEL2770 to occupy the

34

Ser214

  
   

    
       
     

. . _. \
Jar ' -'-

q ‘ ’

'1 a . ‘
0“

§
:- . Km 95
!’ ’l ‘ "p
.4 I: ,\
D 1‘. r
'4'"! I',

A V.

. 9
0‘ ..
..\‘

 

Asp1 89

Figure 2-2/A: Stereoview of the final (2Fo-Fc) electron density of the SEL2711
inhibitor in the thrombin complex. Contoured at the 10 level; SEL2711 is shown in

bold black. Thrombin residues are in thin gray and numbered.

35

\'
b . -
11:35. I Q]; .591 {03" .
.' ’1' .“ " 4i. .... “2
'nl“ ' j ‘5‘" 1.: 0"! 1'33.“ 1‘
l- ‘\\I N." 9:1, ”3.! I: 9 ..‘~'“'.}(_
g g;“' g‘..’.‘. 4“ 'l" ‘.!'.““e‘9 I ".
a '2‘ .1 is: :o. 9.21: '- .1:
H o‘," ‘ ' i“ "‘ 1:75. ’ a f .4 ' .a L"
V \\ I' a. s ’ . 2, “ ‘ K I
w. .t-.~'- ’ ,. M
' a. ‘ .‘I I 1' \‘ _.~Il“' " t“‘=?‘.‘. ' “'.\ ‘1‘“. PI
. ..O\ - .' s “V: éfi‘ "A I ‘$"" 2 g ‘\ (‘8'. V‘?'.'
, . ,5 .. m a,-,,.».- ,. ,\.- , 3 un,‘ .. ya; _.
."'..: ‘0 -r‘ . .“. “ . A,AN‘J.'-‘-r. .2 ‘ (of
4p. . v . ‘9'. he... no, 9,“, i"
‘gyg I I I ‘ E v‘\_9(. .‘ 05.1” . 1’45"!
.7 I I! “if, ,1, .~_ 11V: "' r
‘:I‘ " 7’”! '.4 I’i' $9.‘
,." . "VI,
4'“; I~‘-‘."
1:.‘Qfi ‘9’;
I"?!
1“: ’
V

 

Asp189

Figure 2-2/B: Stereoview of the final (2Fo-Fc) electron density of the SEL2770

inhibitor in the thrombin complex. Contoured at the 10' level; SEL2770 is shown in

bold black. Thrombin residues are in thin gray and numbered.

36

5/ Trp60D a/ Leu99 Hi857
6 O
8\ KT ' lle174 ‘I 1" :2”

l V ' \'(’:.‘\ V I “'\ /€.‘\ ~\QHQS

Cys220
Asp1 89

Figure 2-3: Stereoview of the superimposed Selectide inhibitors in the active site of
thrombin. SEL2711 and SEL2770 are shown in bold black and gray, respectively.

Thrombin residues of the SEL2711 complex are in thin gray and numbered.

37

Table 2-4: Intermolecular and intramolecular interactions in the active site of the
thrombin complexed with SEL2711 and SEL2770 inhibitors. Numbering

and nomenclature as used in text.

 

 

Intermolecular SEL271 l SEL2770 Type
pAphl -O Gly216-N 2.91 2.82 H-bond
-N1 Asp189-OD2 2.89 3.29 H-bond/ion pair
-N2 Asp1 89-OD1 3.07 2.95 H-bond/ion pair
—N1 Ow438/428 3.07 3.04 H-bond
-N2 Gly219-O 3.28 2.62 H-bond
-N2 Alal 90-0 2.87 3.16 H-bond
Phe227-O Ow438/428 3.57 3.16 H-bond
Pal3(Me)3-N Gly216-O 2.69 - H-bond
-N3 Ow455 3.32 - Ion/dipole
les(2Me)3-N Gly216-O - 3 .07 H-bond
-N1 Tyr60A-OH - 3.60 Ion dipole
Leu4-O Ow679 - 2.85 H-bond
Intramolecular
AcO-O les(2Me)3-N1 - 3.43 Ion/dipole
Chg2-O les(2Me)3-N1 - 3.46 Ion/dipole

 

38

proximity of the L-enantiomorphic-S3 site of thrombin similar to the P3 aspartate
residue of a thrombin-thrombin platelet receptor peptide complex (Mathews et al.,
1994). The Chg2 residues of both peptides are in the lower energy chair
conformation; however, they differ in orientation around the CA-CB bond by about
90°. The N-terminal acetyl group of the inhibitors is located in the hydrophobic 82
site of thrombin while the 3-(methyl pyridinium)alanyl residue .(Pal3(Me)3) of
SEL2711 and les(2Me)3 of SEL2770 are in the aryl D-enantiomorphic S3 site; the
pyridyl orientation of the former is approximately orthogonal to the plane of D-
phenylalanyl of PPACK in the thrombin-PPACK complex (Bode et al., 1992). In the
case of SEL2770, the site is occupied by les(2Me)3 in a partially extended
conformation (Figure 2-3). The two C—terminal residues, leucine and proline, point
into solvent region and have no interaction with the thrombin molecule and appear
flexibly disordered in the SEL2711 complex, although they are reasonably fixed in
the SEL2770 complex high B-values notwithstanding (Figures 2-2 & 2-3). This is
somewhat disturbing since crystals of the SEL2711 complex produced a better
diffraction pattern (Table 2-2). Cleavage of the peptide in a substrate binding mode
prior to retro-binding is a possibility but highly unlikely with a Pal3(Me)3 group at the

P1 specificity position (see also below).

IV. DISCUSSION
Because of the structural similarity of SEL2711 and SEL2770, the binding modes

of both are similar (Figure 2-3). While PPACK and many other substrate-like inhibitors

39

show an anti-parallel B—strand association with Ser214-Gly216 of thrombin, SEL271 land
SEL2770 form a parallel B-strand with these residues making hydrogen bonds with the
nitrogen and carbonyl oxygen atoms of Gly2l6 (Table 2-4). The anti-parallel B-strand
association of PPACK in peptidic thrombin inhibition is more abundant because its
hydrogen bonding pattern is energetically preferable compared to that in the parallel B-
strand alignment of SEL2711 and SEL2770. The parallel B-strand arrangement, however,
displays a high degree of fidelity in its positioning in the active site when compared
among other known retro-bindin g thrombin inhibitors.

The largest structural difference in the binding of the two similar inhibitors with
thrombin appears at the S3 site of the enzyme where the more flexible N1ys(2Me)3 group
of SEL2770 is able to adopt a favorable conformation to interact internally with itself and
thrombin (Figure 2-3). Superposition of the two inhibited structures shows that the
quaternary nitrogen atoms of N1ys(2Me)3 and Pal3(Me)3 are 3.3 A apart. However, in
the case of the former, free bond rotations place the positively charged nitrogen atom in
an electronegative environment consisting of the carbonyl oxygen atoms of the acetyl
group and Chg2O of SEL2770, as well as Tyr60A-OH of thrombin (Table 2-4). If the
N1ys(2Me)3 group were fully extended, it could approach the carbonyl oxygen atom of
G1u97A within 3.0 A. Thus, the ion-dipole interactions of SEL2770 appear to be more
stabilizing than this possible hydrogen bond. With much less rotational freedom, the
positively charged nitrogen atom of Pal3(Me)3 cannot orient in a direction toward the
same internal electronegative environment utilized by N1ys(2Me)3 and thus achieve a
comparable interaction (Pal3(Me)3-N3 to Ac0—Ol = 5.0A; to Chg2-O, 5.1A; to Tyr60A-

OH, 4.7A; compare with corresponding N1ys(2Me)3-N1 distances in Table 2-4).

40

The Chg2 residues of both inhibitors occupy the L-enantionmorphic-S3 region of
thrombin similar to aspartate of Leu-Asp—Pro-Arg in a thrombin-thrombin platelet
receptor peptide complex (Mathews et al., 1994). The L—S3 site is surface exposed and of
considerable size (Figure 2-3): the Chg2 residues do not superpose on the aspartate
position of the former complex but rather are located adjacent to it and even appear to be
in approximately orthogonal orientations (Figure 2-3) (x1 = 173° and 71° for SEL2711 .
and SEL2770, respectively). Both the Chg2 groups of SEL2711 and 2770 and the
aspartate group of thrombin platelet receptor peptide point into solvent space. The
aspartate, however, makes a water mediated hydrogen bond with Arg22lA to stabilize its
orientation (Ni et al., 1992; Mathews et al., 1994), while the Chg2 groups cover the 81
site and appear to interact with phenyl pAphl group in the specificin site and the
disulfide bridge of Cysl9l-Cy3220. Thus, the Chg2 groups assume the usual role of
Glul92 (bent conformation covering 81) and in the process make close contacts with
Cysl9l-S and Cy8220-S providing a more hydrophobic environment for the latter by
expelling some water molecules from the region.

The different backbone associations with thrombin notwithstanding, PPACK and
the Selectide inhibitors display similar binding within the 81 specificity pocket. In both
structures, two nitrogen atoms of the guanidino group (PPACK) and the arnidino group
(SEL2711 and SEL2770) form a doubly hydrogen bonded salt bridge with the carboxylate
of Asp189. In addition, one of the nitrogen atoms also interacts with Gly219-O and the
other makes a water mediated bridge to Phe227-O. Two more water molecules, one

bridging from Asp189-OD2 to Tyr228-OH and the other bound to Asp189-OD1, both

41

also present in the apo-thrombin structure, remain in the specificity pocket on binding of
the inhibitors. In the thrombin-PPACK structure, an additional water molecule in the
specificity pocket mediates an interaction between the arginyl NE atom and Glul92-0E1
while in SEL2711 and SEL2770, this interaction is lost because Glul92 is in the
thrombin-hirugen conformation (extended) (Vijayalakshmi et al., 1994). The bulkiness of
the incoming aromatic ring of pAphl or that of Chg2 appears to disrupt the usual
conformation of Glul92 (bent over the 31 site) that accompanies arginyl binding at the
specificity site. Residue Glul92 adopts a fully extended open conformation and does not
have any specific contacts, being positioned on the surface of thrombin, which allows the
P2 Chg2 group of the inhibitors to occupy the same site as the P3 aspartate of a thrombin-
thrombin platelet receptor peptide complex.

Superposition of the Selectide inhibitors and the thrombin retro-binding BMS—
183507 inhibitor (PDB code: T5161) reveals a practically identical parallel B-strand
association of the backbone of the inhibitors with the Ser214-Gly216 residues of
thrombin (Figure 2-4). Like PPACK, BMS-183507 forms three hydrogen bonds with
Ser214-O, Gly2l6-N and Gly216-O of thrombin, while the Selectide inhibitors have only
two (Table 2-4). The additional hydrogen bond of EMS-183507 is accomplished by the
different positioning of the terminal amide group. The arginyl-like group of the BMS
inhibitor has one carbon atom less than an arginyl side chain, so its entrance to the

specificity site leads to a bowed conformation that positions the terminal amide within

42

/
e“ e I His57
t» ‘x ("o‘x

’ l/lq‘) y ./"’\I?,“ M195
«9 a
I e 4

\<\(\ Cys220 tr
Asp1 89

Figure 2-4: Stereoview of superimposed SEL2711 and BMS-183507 bound to
thrombin. SEL2711 and BMS-183507 are shown in bold black and gray, respectively.

Thrombin residues of the SEL2711 complex are in thin gray and numbered.

43

hydrogen bonding distance of Ser214-O (Figure 2-4). The N-terminal arginyl—like moiety
and phenyl group of BMS-183507 bind in the specificity pocket and 82 site, respectively,
whereas the 81 site is occupied by the Aphl group of the Selectide inhibitors while the N -
terminal acetyl group is in the 82 position (Figure 2-4). In addition, the BMS and the
Selectide inhibitors display different binding modes within the 81 site. Only one of the
nitrogen atoms of the guanidine of BMS-183507 makes contact with a carboxylate
oxygen atom Of Asp189 while the other hydrogen bonds to Gly219-O like the Selectide
inhibitors (Figure 2-4; Table 2-4).

Since the structures of the catalytic domains of FXa (Padmanabhan et al., 1993)
and FVHa (Banner et al., 1996; Zhang et al., 1999) are similar to that of thrombin (rmsA
= 1.2 A and 1.1 A, respectively), which especially applies to the active site regions, it is
quite possible that the observed retro-binding mode of the Selectide inhibitors with
thrombin may also be operative with FXa and FVHa. This possibly was explored by
transposing the thrombin bound Selectide inhibitors on the active sites of FXa and FVHa.
(Figures 2-5 & 2-6). The principal sequence differences in the resides surrounding the
81-83 binding subsites of the two enzymes are: Leu99, Ile174 and Glul92 of thrombin
are replaced by Tyr99, Phel74 and Glnl92 in FXa and by Thr99, Prol701 and Lysl92 in
FV Ha, respectively. An even larger difference is the absence in FXa and FVHa of the 60-
insertion loop of thrombin. The Tyr60A - Trp60D segment of the loop projects out over
the 82 and D-enantiomorphic S3 binding sites in thrombin restricting their access and
thus contributing to specificity.

Selectide inhibitors modeled into the active site of FXa: Theoretical

considerations have led to the proposal of cation-1t electron mediated interactions

 

Figure 2-5: Stereoview of SEL2711 and SEL2770 superimposed on the active site of
FXa in a retro-binding mode. SEL2711 and SEL2770 are shown in bold black and
gray, respectively. FXa residues are in thin gray and numbered. Since the pyridine of
SEL2770 collides with Phel74 of FXa, a conformational change must occur in one or

the other, most likely in the former.

45

Th r99

Pr0170|
C‘ ‘7 G, r i .i’ 9' " F557
1 , l

. '4
‘ ’\ Ser214

” ) Ser195

/ I

Asp1 89

I.

T1
(3. .

Figure 2-6: Stereoview of SEL2711 and SEL2770 superimposed on the active site of

W in a retro-binding mode. SEL2711 and SEL2770 are shown in bold black and gray,

respectively. FVHa residues are in thin gray and numbered.

46

(Dougherty & Stauffer, 1990) in the S4 binding site of FXa (Lin & Johnson, 1995) where
the n-faces of the three aromatic residues Tyr99, Phel74 and Trp215 are sufficiently rich
in it electrons that they not only form a hydrophobic pocket but also act as a cation
recognition site. This region also corresponds to the D-enantiomorphic S3 site, which is
occupied by D-Phe in the thrombin-PPACK complex. The P4, L-leucine residue of the
thrombin-LDPR complex of thrombin platelet receptor peptide also occupies this site in
thrombin (Mathews et al., 1994).

Thus, the charge interaction is basically an attraction of a positive charge to the 71:-
electrons of the aromatic rings, alternatively called an ion-quadrupole attraction (Stauffer
et al., 1990; Schwabacher et al., 1993). The cation-1t electron binding hypothesis was
modified somewhat with the structure determination of the DX-9065a (Daiichi
compound) inhibited structure of FXa (Brandstetter et al., 1996), which showed that the
above aryl-binding site was further assisted by the carbonyl oxygen atoms of Lys96-
Glu97 and the side chain of the latter to form a so-called cation hole that expands the S4
binding site region. It is noteworthy that the main chain of FXa between 94 - 98 is
positioned differently in thrombin due to an insertion in the vicinity of G1u97A of the
latter. In the absence of the insertion, Lys96 - Glu97 of FXa make closer contacts with
groups binding at the D-S3 site. It is also note that the Arg93 - ArglOl stretch has been
implicated in thrombin heparin binding by electrostatic potential energy calculations
(Karshikov et al., 1992) and by mutagenesis (Gan et al., 1994; Ye et al., 1994; Sheehan et
al., 1994). Thus, under appropriate circumstances, the two sites (S4 and the heparin

binding exosite H) could interact with each other.

47

If the Selectide inhibitors bind to FXa in a similar retro-manner as with thrombin,
the positively charged Pal3(Me)3 and N1ys(2Me)3 groups occupy the aryl site formed by
Tyr99, Phel74 and Trp215 (Figure 2-5). In the case of SEL2711, the Pal3(Me)3 group,
modeled as it binds to thrombin, makes edge-to-face interactions with Tyr99 and Phel74.
The latter aromatic ring systems are both parallel to the pyrrolidine ring of the inhibitor in
the DX-9065a-FXa complex (Brandstetter et al., 1996). The different orientations could
reflect the difference between an aromatic and aliphatic group interacting with the aryl
site of FXa or it could reflect a more accurate determination of orientation of the
Pa13(Me)3 group (2.1 A resolution) compared to the pyrrolidine (3.0 A resolution). If no
conformational changes occur to FXa accompanying binding, the positively charged
nitrogen atoms of the two Selectide inhibitors are too far away (> 4.5 A) to hydrogen
bond with the carbonyl oxygen atoms of Lys96 - Glu97 of the S4 cation hole. The three
residues of the aryl site, however, can interact with the delocalized positive charge of the
Pal3(Me)3 ring similar to that described for the Daiichi compound (Brandstetter et al.,
1996) to approximate a cation-1t electron mediated ion-quadrupole attraction (Lin &
Johnson, 1995). Since the binding constants of the two Selectide inhibitors are
practically the same for FXa (Table 2-1), the N1ys(2Me)3 group most likely achieves a
similar cation - 1t electron mediated interaction as the Pal3(Me)3 group of SEL2711.

Conversely, if the Selectide inhibitors bind to FXa in a substrate-like binding
mode, the arnidino nitrogen atoms of the pAphl groups of both inhibitors could form
hydrogen bonds with Lys96-O and Glu97-O of the S4 cation hole; however, the binding
of the Pal3(Me)3 and N1ys(2Me)3 groups in the S1 specificity site is clearly inferior to

that of the doubly hydrogen bonded salt bridge of pAphl in retro-binding. Thus, a retro-

48

binding mode in FXa would appear to be more preferable. If the inhibitors bind in a
retro-manner in both thrombin and FXa, the difference of a factor of 1.3 x 104 (SEL2711)
and 4 x 103 (SEL2770) in selectivity (Table 2-1) would be due to the contribution of the
Pal3(Me)3 and N1ys(2Me)3 groups binding at the S4 - S3 cation-aryl site of FXa. This
only corresponds to 5 - 6 kcal mol'1 and from all appearances, the site can either operate
through cation-1t electron mediated interactions (Lin & Johnson, 1995) or with a longer
inhibitor group to reach Lys96 - Glu97, hydrogen bonding could also occur in the cation
hole (Brandstetter et al., 1996).

Selectide inhibitors modeled into the active site of F VIIa: If the Selectide
inhibitors bind to FVHa in a retro-manner, as they do in thrombin, and if there are no
protein conformational changes accompanying binding, the inhibitors retain their
intermolecular interactions in the specificity pocket as well as with Gly216-O (Figure 2-
6). However, the longer ion-dipole interaction between positively charged Pal3(Me)3-N 3
(SEL2711) or N1ys(2Me)3-N 1 (SEL2770) with Tyr60A-OH is lost due to the absence of
the 60-insertion loop of thrombin. While in FXa this loss is compensated through cation—
tt electron mediated interactions at the aryl site formed by Tyr99, Phel74 and Trp215
(Figure 2-5) which result in a significant increase of selectivity of the inhibitors for FXa
(Table 2-1), in FVHa this loss remains uncompensated. The residues Thr99 and Prol701
of FVHa, which are structurally equivalent to Tyr99 and Phel74 of the aryl site of FXa,
form neither a hydrophobic pocket nor a cation recognition site. These loss of
interactions results in weaker binding of the Selectide inhibitors to FVHa: the differences
are a factor >5 (SEL2711) and >25 (SEL2770) with respect to thrombin (Table 2-1). The

difference of five between these two difference arises from the closer contact observed in

49

SEL2770, between N1ys(2Me)3-N1 and Tyr60A-OH (3.6A), while Pal3(Me)3-N3 and
Tyr60A-OH are separated by 4.7A in the SEL2711 complex.

Binding of Peptides A and B to the active site of FXa: Two other inhibitors
developed by Selectide Corp. (peptides A and B mentioned earlier), have been shown by
using transferred nuclear Overhauser effect NMR spectroscopy (Fratemali et al., 1998) to
have a very compact conformation displaying a severe hydrophobic collapse of the P2 —
P3 groups in the substrate binding site of FXa and not resembling the usual substrate .
binding mode. The arginine of the peptides enters the specificity 81 site differently from
substrate, in an extended but curved conformation, surprisingly forming a doubly
hydrogen bonded salt bridge with Asp189, while the P2 and P3 residues (Ile-Tyr for
peptide A, Chg-4-arnino-Phe for peptide B), which must be the source of the selectivity
for FXa, interact together above the 81 site with the 82 site and Tyr99. These bound
structures are very different from the retro-binding mode modeled here for SEL2711 and
SEL2770 where selectivity for FXa appears to come from a cationic interaction with the
S3 — S4 sites. The only significant factor that would seem to underlie the binding
difference thus appears to center about the 81 specificity site. It has already been
indicated that the Pal3(Me)3 and N1ys(2Me)3 groups of SEL2711 and SEL2770 are
inferior P1 residues compared to the pAphl group and is most likely the reason why these
inhibitors do not bind like peptides A and B. This is also generally in agreement with the
weaker binding constants of peptides A and B for FXa, being only 1.6 and 0.3 11M,
respectively, which essentially result from side chain-side chain hydrogen bond
interactions between inhibitor and enzyme. Thus, three or more different active site

binding modes (including anti-parallel B-stand substrate binding) may be operative in

50

FXa depending on the exact structure of the inhibitor, the selectivity of which is derived

from interactions with the 84 region in the retro-binding case.

51

CHAPTER 3
SELECTIVE NON-ELECTROPHILIC INHIBITORS OF

THROMBIN

I. MOLECUMETICS INHIBITORS

Peptidic thrombin active site inhibitor can be broadly classified under two types,
electrophlic and non-electrophlic, according to their ability to directly interact with
Ser195-OG of the catalytic triad of thrombin. Electrophilic inhibitors, such as the widely
studied model ligand PPACK, form a covalent tetrahedral intermediate between an
activated carbonyl group of the Pl residue of the inhibitor and Serl95-OG, while all
interactions between thrombin and non-electrophlic inhibitors are strictly non—covalent.
Since structural similarity of the catalytic centers of thrombin and other serine proteases
that also play important physiological roles in life processes, electrophilic inhibitors can
operate with a very large array of serine proteases without much selectivity, causing
unwanted side effects. On the other hand, non-electrophilic inhibition of thrombin is
highly sensitive to the geometry and charge distribution of the active site of the enzyme as
well as structural elements of the inhibitors that all lead to greater discriminative
inhibition between thrombin and other serine proteases.

With this notion, Molecumetics Ltd. (Bellevue, WA) has used a combinational
chemistry approach to generate a diverse variety of non-electrophlic conforrnationally-

constrained mimetics of the principal structural elements of proteins (a-helicies, [3-

52

strands, reverse turns) (Kahn et al., 1988; Nakanishi et al., 1992; Eguchi et al., 1996; Kim
& Kahn, 1997). The syntheses (Boatman et al., 1999) and the crystal structures (St.
Charles et al., 1999) of four Molecumetics (MOL) thrombin active site inhibitors bound
to the enzyme were recently reported and classified as first generation of (P3—P1') B-
strand templated thrombin inhibitors with the hetero-bicyclic P2 group. The binding
mode of these inhibitors is similar to that of PPACK: the bicyclic and phenylalanyl
groups of each inhibitor approximate the extended conformation of PPACK in PPACK-
bound thrombin (St. Charles et al., 1999).

The second generation of MOL inhibitors (Figure 3-1) has an amino, arnidino or
amino-irnidazole group attached to a 1,4-substituted cyclohexyl ring at the P1 position
that can simulate arginine- or lysine-like binding at the specificity pocket of thrombin. A
modified, fused three-nitrogen, hetero-bicyclic ring (Bic) constitutes the central core of
the inhibitors expected to associate with the Ser214-Gly216 stretch of thrombin in the
antiparallel B-strand manner. The P3 benzyl (MOL354, MOL356, MOL376 and
MOL592) or bromobenzyl (MOL1245) group is designed to occupy the D-
enantiomorphic-S3 binding site of thrombin. From analysis of the binding constants, the
second generation of MOL inhibitors possesses more optimal binding features at the Sl-
S3 sites that result in highly potent and selective inhibition of thrombin. The three best
thrombin inhibitors (MOL354, MOL1245 and MOL376) are also the most selective (2-3

orders of magnitude) against trypsin (Figlre 3-1).

53

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54

II. EXPERIMENTAL

Crystallization of the MOL3 76 and MOL592 complexes]: A 7-fold molar excess
of hirugen was added at 4°C to a frozen human a-thrombin solution (1.0 mg/mL in 0.75
M NaCl) to form a 1:1 complex and prevent autocleavage of the enzyme. In
approximately 24 hours, the solution of the formed binary thrombin-hirugen complex was
concentrated in a refrigerated centrifuge (4°C) to about 7.0 mg/mL using a Centricon 10
concentrator with a molecular weight cutoff of 30 kDa. This concentrated thrombin-i
hirugen solution were left overnight before MOL376 and MOL592 were added to the
thrombin-hirugen complex in a 50 to 60-fold molar excess.

The ternary complexes were crystallized using the hanging drop crystallization
technique in a 2 it] drop, which consisted of 1 11L of the protein sample and l ”L of a well
solution. The well solution contained 24% PEG 8000 in 0.1 M SPB, pH 7.3. The
repetitive macroseeding technique was applied to enlarge the crystals to the size of 0.2 x
0.2 x 0.1 mm (MOL376) and 0.3 x 0.2 x 0.2 mm (MOL592).

Intensity Data Collection: The X-ray diffraction data of the thrombin-hirugen
complexes inhibited with MOL376 and MOL592 were collected at —150 °C with a R-
AXIS II imaging plate detector using 30% PEG 4000, 15% glycerol in 50 mM SPB, pH

7.3 as cryo-solvent. The radiation generated from a Rigaku RU200 rotating anode

 

1 Crystal structure and the binding modes of MOL376 and MOL592 to thrombin
presented in this chapter are the part of larger study that includes structure determination
and comparative analysis of the binding modes of the MOL354, MOL356 and MOL1245

complexes.

55

operating at 5 kW power with the fine focus filament (0.3 x 3.0 mm) was monochromated
(CuKa) and intensified by focusing with Molecular Structure Corporation - Yale mirrors.
The crystal to detector distance was 10.0 cm with a swing angle of zero degrees.
Autoindexing and processing of the measured intensity data were carried out with the
Rigaku R-AXIS software program package (Higashi, 1990). Characteristics of the
MOL376 and MOL592 crystals and the intensity data collection statistics are given in
Table 3-1.

Structure Determination: The MOL376- and MOL592-thrombin-hirugen crystals
belong to the monoclinic space group C2 with four ternary complexes per unit cell. Their
crystal structures were determined directly using the isomorphous thrombin coordinates
of the binary thrombin-hirugen complex (Vijayalakshmi et al., 1994; PDB code: 1HAH).
The initial coordinates were optimized by rigid-body rotation-translation refinement in
the (9.0 - 3.0) A resolution range using the X-PLOR program package (Brunger, 1992).
The structures were refined with the programs PROLSQ (Hendrickson, 1985) and
PROFFT (Finzel, 1987) generally following procedures described in Chapter 2. The final

refinement statistics of the complexes are presented in Table 3-2 and 3-3.

III. RESULTS

Thrombin: Electron density is well defined for most of the residues of thrombin in
both MOL the complexes, except for the terminal regions of the A chain (ThrlH - AlalB,
Ilel4K - Arng) and the autolysis loop in the vicinity of Trp148 - Lysl49E, both of which

are also typically disordered in most other isomorphous thrombin structures. The

56

Table 3-1: Intensity data collection of Molecumetics inhibitors MOL376 and

 

 

MOL592 bound to thrombin.
MOL3 76 MOL592
Space group C2 C2
Cell constants
a (A) 71.46 71.43
b (A) 72.19 71.67
c (A) 73.12 72.45
B(deg) 100.7 101.0
Complexes/asymmetric unit 1 1
Resolution (A) 2.3 2.0
Observations 14,987 33,066
Outermost range (A) 2.5-2.3 2.2-2.0
R-merge (%) 5.8 5.7
Outermost range 13.1 15.8
Independent reflections 9,294 17,371
Redundancy l .6 1.9
Completeness (%) 56 72
Outermost range 38 42
I/o(I) (outermost range) 2.6 2.8

 

57

Table 3-2: Refinement summary of deviations from ideality of thrombin molecules

in the MOL376 and MOL592 complexes.

 

 

Restraints Target MOL376 MOL592

Distances (A):

Bond distance 0.020 0.011 0.014

Angle distance 0.030 0.037 0.036

Planar 1,4 distance 0.050 0.047 0.046

Planes 0.040 0.034 0.030
Chiral volume (A3) 0.15 0.17 0.17
Non-bonded contacts (A)

Single-torsion 0.60 0.22 0.24

Multiple torsion 0.60 0.25 0.28

Possible H-bond 0.60 0.25 0.28
Isotropic thermal factors (A2)

Main-chain bond 1.0 1.0 0.9

Main-chain angle 1.5 1.7 1.5

Side-chain bond 2.0 2.5 2.1

Side-chain angle 3.0 3.6 3 .0

 

58

Table 3-3: Individual refinement statistics of MOL376 and MOL592 inhibitors.

 

 

MOL376 MOL592
Resolution range (A) 9.0-2.3 9.0-2.0
No. of reflections 9092 17152
IFol/o > i 1.5 1.5
R-value (%) 15.3 17.4
No. of water molecules 98 134
<B> (A2) 28.4 28.3

 

final thrombin structures of the ternary complexes are similar and show no significant
conformational differences from each other as well as other thrombin structures (rmsA <
0.4A between 240-250 CA positions). The Ramachandran plots (Laskowsky et al., 1993)
of the MOL376 and MOL592 complexes show that 191 (78.9 %) and 200 (82.3 %)
residues of thrombin are in most favorable regions while 50 (20.7 %) and 43 (17.7 %)
residues occupy additionally allowed areas (Figure 3—2). There is no reside in the
disallowed regions and only one residue (MOL356) is in the generously allowed region.
Two Na+ ion sites have been found in the monoclinic crystal structures like those initially
described (DiCera et al., 1995; Zhang & Tulinsky, 1997). Both sodium ions adopt
slightly distorted octahedral arrangements coordinated by solvent water molecules and

carbonyl oxygen atoms: those of Arg221A and Lys224 for the intramolecular

59

 

Ramachandran Plot
MOL376

 

Psi (degrees)
C:

-45
-90 '
- l 35
_1 80 . l -.
-1 8O -1 35 ‘ 45 ' 0
Phi (degrees)
Plot statistics
Residues in most favoured regions [A.B.L] 191 78.9%
Residues in additional allowed region! [a.bJ.Pl 50 20.7%
idues in generously allowed region: l—a.~b.-l.-p] l 0.4%
Residues in disallowed regions 0 0.0%
Number of non-glycine and non-proline residues 242 1111M
Number of end-residues (excl. Gly and Pro) 8
Number of glycine residues (shown :3 triangles) 23
Number of proline residues 16
Total number of residues 289

 

 

 

Figure 3-2/A: The Ramachandran plot of the final structure of the thrombin-MOL376

complex. Glycine residues are shown as triangles. Non-glycine residues as squared

boxes.

60

 

Ramachandran Plot
MOL592

 

Psi (degrees)

 

 

 

45
Phi (degrees)
Plot statistics
Residues in most favoured region: [A. B L] 200 82.3%
Residues in additional allowed regions [a.bl .p] 43 17.7%
Residues in generously allowed regions l-a.- -.—b —l-pl 0 0.091
Resrdues in disallowed regions 0 00‘}
Number of non-glycme and non-proline residues 743 [(00%
Number of end- rendues (excl Gly and Pm) 7
Number of glycine residues (shown as triangles) 22
Number of proline resid net 16
Total number of residues 288

 

 

 

Figure 3-2/B: The Ramachandran plot of the final structure of the thrombin-MOL592

complex. Glycine residues are shown as triangles. Non-glycine residues as squared

boxes.

61

site while those of Lysl69 and Thrl73, as well as Phe204A from a symmetry related
molecule, coordinate the intermolecular ion (Zhang & Tulinsky, 1997). The occupancies
and the temperature factors of the Na+ ions of the MOL376 and MOL592 complexes are:
9:0.97, 13:26.9A2 and (2:098, B=27.8A2 (intramolecular) and (2:0.67, B=28.6A2 and
Q=0.95, B=3 1 .0A2 (intermolecular), respectively.

Molecumetics inhibitors: The MOL376 and MOL592 complexes have been
refined at a resolution of 2.3 A (MOL376) and 2.0 A (MOL592) (Tables 3-1 & 3-2). The
electron density of both inhibitors is well defined for all residues (Figure 3-3). Unlike
SEL2711 and SEL2770, MOL376 and MOL592 bind to thrombin like a substrate or the
archetype inhibitor PPACK (Bode et al., 1992) forming an antiparallel B-strand with
residues Ser214-Gly216 of thrombin. The cyclohexyl amino (MOL376) and cyclohexyl
amino-imidazole (MOL592) P1 groups occupy the specificity pocket and make one and
two direct interactions, respectively, with the negatively charged carboxylate group of
Asp189. The P2 hetero-bicyclic Bic groups are practically planar with some small
deviations from planarity in the six-membered ring. The Bic group of each inhibitor
occupies the apolar S2 and part of the 83 region of the active site making hydrophobic
contacts with the side chains of Hi357, Tyr60A, Trp60D and Trp215 like the first
generation of bicyclic groups (St. Charles et al., 1999). The six-membered ring occupies
approximately the same position as the proline of PPACK in thrombin-PPACK (Bode et
al., 1992). Bridged by the sulfonyl, the P3 benzyl groups of MOL376 and MOL592 curl

up toward the 60-insertion loop of thrombin and make numerous hydrophobic

62

LySBOF

Trp60D

 

Glu97A

Hi857

Ser1 95

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Asp1 89

Figure 3-3/A: Stereoview of the final (2Fo-Fc) electron density of the MOL376 inhibitor

Contoured at the 10' level. MOL376 is in bold black.

in the thrombin complex.

Thrombin residues are in thin gray and numbered.

63

    

GIu97A

’e
.‘ .
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Glu217

Figure 3-3/B: Stereoview of the final (2Fo-Fc) electron density of the MOL592 inhibitor
in the thrombin complex. Contoured at the 10' level. MOL592 is in bold black.

Thrombin residues are in thin gray and numbered.

interactions with thrombin residues Asn98-Leu99, Ilel74 and Trp215 of the D-
enantiomorphic-S3 binding site. The orientation of the benzyl ring of both inhibitors is
approximately orthogonal to the plane of the indole ring of Trp215, in a fashion typical of

aromatic-aromatic interactions in proteins (Figure 3-3).

W. Discussron

As mentioned earlier, MOL376 and MOL592 together with MOL354, MOL356
and MOL1245 belong to a second generation of conformationally-constrained
Molecumetics inhibitors (Figure 3-1). Because of the close structural similarity of these
inhibitors, the overall binding modes are similar and relate to that observed in the PPACK
complex (Bode et al., 1992). The amino, amidino or amino-imidazole charged group
attached to a cyclohexyl ring at the P1 position form hydrogen bonded salt bridges with
the negatively charged carboxylate of the Asp189 side chain (Figures 3-4 & 3-5).
Differences in the chemical nature of the charged basic groups, however, and the overall
size and exact conformation of the P1 groups produce variations in binding interactions
and the role of solvent in the stabilization of the thrombin-MOL inhibitor complexes
(Figure 3-6). The cyclohexyl group of MOL376, like those in MOL354 and MOL1245,
appears to be in the energetically favored chair conformation (Figure 3-4); however, in
MOL592 and MOL356 the cyclohexyl ring is in a boat conformation (Figure 3-5).

The cyclohexylamino group of MOL376 interacts with Asp189 of thrombin more
like the lysine residue of the first generation MOL106 inhibitor (St. Charles et al., 1999)

rather than the lysine analogue of DUP714 (Weber et al., 1995), where a water molecule

65

 

"6174
Gly216

 

Asp189

Figure 3—4: Stereoview of superimposed MOL354/376/ 1245 bound to thrombin.
MOL354 is in bold black; MOL376 is broken black line; MOL1245 is in bold gray.

Thrombin residues of the MOL1245 complex are in thin gray and numbered.

66

 

Glu217

Asp189

Figure 3-5: Stereoview of superimposed MOL356 and MOL592 bound to thrombin.
MOL356 is in bold black; MOL592 is in bold gray. Thrombin residues of the MOL356

complex are in thin gray and numbered.

67

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68

bridges the lysine amine and the Asp189 carboxylate group. This is most likely because
the similarity of the large hydrophobic bicyclic P2 groups of MOL106 and MOL376
which align the inhibitors in the active site of thrombin in a similar orientation. In both
MOL complexes, the amino group makes a hydrogen bonded salt bridge with Asp189-
ODl (Figures 3—4 & 3-6). Moreover, in the MOL376 complex, the bond to the
cyclohexyl ring is equatorial and an equatorial cyclohexylamino group occupies a
position equivalent to one of the terminal nitrogen atoms of the guanidinum group of
arginine- or arnidino-based inhibitors (Figures 3-4 & 3-6). Other conserved interactions
of such inhibitors are also observed in the 81 site (Zhang & Tulinsky, 1997), which are a
hydrogen bond to Gly219-O and a doubly hydrogen bonded water molecule mediating
Asp189-OD2 and Tyr228-OH (Figure 3-6). Another water molecule, Ow603 positionally
mimics a NH] or NH2 atom of thrombin-bound arginine and completes the interactions
of the amino group bridging between it and Asp189-OD2 (Figure 3-6). The second
solvent shell of MOL376 includes the conserved water site of Ow426 hydrogen bonded to
Phe227-O and 0,603. Although Aspl89-OBI of thrombin and the cyclohexylamine of
MOL376 hydrogen bond to a water molecule (Ow605) as in thrombin complexes with
arginine-based inhibitors, unlike the latter, no further interactions ensue from this water
because it is positionally a little different. Some of the foregoing interactions have also
been observed with other thrombin bound inhibitors containing a cyclohexylamine or an
aminopyridine group at the Pl position (Tucker et al., 1997; Feng et al., 1997).

Both MOL356 and MOL592 have cyclohexyl-aminoimidazole groups occupying
the 81 site but since the amide nitrogen lacks a methylene carbon, it is linked directly to

the cyclohexyl ring so the latter does not penetrate as deep into the 81 site and only

69

overlaps about half of the cyclohexyl ring of MOL354, MOL376 or MOL1245. The
cyclohexyl rings of MOL356 and MOL592 are in the boat conformation and the
stereochemistry of the bonds to the cyclohexyl and imidazole groups of the two (both
apical positions) are the same being axial and equatorial, respectively; however, the
interactions of the amino-imidazole groups are very different in the specificity pocket
(Figures 3-5 & 3-6). The imidazole ring is parallel to the plane of the boat in MOL592
whereas it is rotated about 75° in MOL356. Moreover, the MOL592 inhibitor forms a
doubly hydrogen bonded salt bridge with Asp189, through a nitrogen atom of the
imidazole ring and its amino group. This is unlike MOL356, which interacts with
Asp189 uniquely (Figures 3-5 & 3-6). Thus, the terminal imidazole of the P1 group of
MOL592 simulates more a guanidinium of arginine utilizing all three nitrogen atoms
(Figure 3-6). This amino-imidazole mimics arginine further by having other conserved
hydrogen bonded interactions found in thrombin-arginine complexes (Krishnan et al.,
1998). The imidazole ring of MOL356 is flipped 180° with respect to MOL592 about its
bond to the cyclohexyl ring and only one imidazole nitrogen atom forms a hydrogen bond
with Asp189-OD2 while the amino nitrogen makes a direct hydrogen bonding contact
with Phe227-0 occupying the position of a conserved water molecule of other complexes
(Figure 3-6). This water molecule is linked to the charged terminal guanidinum or
amidine group by a hydrogen bond in P1 arginyl or amidino inhibitors.

The 01 oxygen atoms of the Bic groups of the inhibitors form a hydrogen bond
with Gly216-N like the bicyclic P2 group of the first generation Molecumetics inhibitors
(St. Charles et al., 1999). This interaction is in all the thrombin-MOL structures ( < 3.0

A) and corresponds to one of the three usually found in the antiparallel B-strand between

70

P2-P3 residues of inhibitor and Ser214-N, Gly216-N and Gly216-O. The usually
accompanying interaction from P3 nitrogen to Gly216-O, which is observed in the
SEL2711 and SEL2770 complexes (Table 2-3), is missing with the present Molecumetics
inhibitors because they lack the nitrogen atom. A slightly longer hydrogen bond contact,
which varies between (3.1 — 3.3) A, in the antiparallel B-stretch is found between the
amide nitrogen of the MOL354/376/ 1245 inhibitors and Ser214-O. This interaction is
observed-in thrombin complexed with PPACK (substrate binding mode) and with BMS-
183507 (retro-binding mode).

The sulfonyl groups of MOL376 and MOL592 are located on the surface and have
no interactions with thrombin. This is in agreement with other P3 benzylsulfonyl
inhibitors bound to thrombin (Krishnan et al., 1998). In comparable complexes with
trypsin, the benzyl group is disordered and the sulfonyl is positionally displaced and
interacts with Gly218—N through a hydrogen bond (Krishnan et al., 1998). A similar
hydrogen bond has been reported with thrombin and two benzylsulfonarnide inhibitors
(Tucker et al., 1997; Feng et al., 1997). However, these particular inhibitors have a D-
Phe at P3 that occupies P3, with the benzylsulfonated N-terminus of the inhibitor. The
benzyl group thus occupies a uniquely different position close to a P1 cyclohexyl or
pyridylamine.

The crystallographic determination of the binding modes of the Molecumetics
inhibitors is used here to correlate the trends in the binding constants of the inhibitors for
thrombin. The two best inhibitors for thrombin are MOL354 and MOL1245, which have
a cyclohexylamidino P1 group and sub-nanomolar binding constants (Figure 3-1). Based

on the binding constants, the arnidino group of MOL354 or MOL1245 mirnicks the

71

guanidinium of arginine and substantially contributes to the binding constant, while the
contribution of the P3 groups should be comparable. The majority of the loss of a factor
of about 20-30 in thrombin binding of MOL376 is clearly the result of the replacement of
the amidino with an amine group at P1. Although the amine of MOL376 copies arnidino
binding to some extent (Figure 3-6), the overall energetics of binding appears to be
weakened. The cyclohexyl-aminoimidazole inhibitors are the poorest inhibitors of
thrombin (Figure 3-1), which must be due to the bulkiness of their P1 group. rather than
delocalization of the positive charge within the imidazole ring. It is difficult, however, to
account for the markedly poorer binding constants of MOL356 and MOL592 from their
specificity site interactions (Figure 3-6) that suggest more or less comparable binding to
the other Molecumetics inhibitors. MOL356 is a better inhibitor of thrombin than
MOL592 by two orders of magnitude. It is most likely due to a larger number of direct
interactions of MOL356 with thrombin, rather than a larger number of water mediated

ones observed in MOL592 (Figure 3-6).

72

CHAPTER 4:

PROBING THE ACTIVE SITE OF THROMBIN FURTHER WITH UREA

I. INTRODUCTION

A way to determine relationships between tertiary structure and biological activity
of enzymes is to alter the structure and study the effect of the alternation on the biological
functions of the enzyme. Structure alternations in proteins caused by heat, extreme pH,
or by exposure to organic solvents, detergents or certain solutes are referred to as
denaturation. The structural changes caused by denaturation are generally associated
with loss of catalytic activity and are a consequence of catalytic fianctions depending on
its specific three—dimensional structure. The process of denaturation is usually reversible:
most of the denaturated proteins will regain their native structure and, therefore,
biological activity, if they are returned to a condition in which the native conformation is
stable. This phenomenon of reversibility is one of the proofs that the tertiary structure of
proteins is determined by amino acid sequences and not by the path by which a protein
folds into its native conformation. Previous studies of the effect of denaturating agents
on the biological properties of various proteins may be broadly classified into two
categories: 1) those that examine dependence of the protein activity as a function of
concentration of denaturating agents and 2) those that examine denaturant binding sites in
the protein at the molecular level.

The effect of denaturating agents on biological activity of protein is a multi-
facetted subject. Many enzymes with covalently intact structures exhibit sigmoidal,

almost fiilly reversible unfolding upon treatment with denaturants in non-reducing

73

conditions. These enzymes include widely used blood proteins such as trypsin (Harris,
1956), a-chymotrypsin (Martin & Frazier, 1963) and (it-thrombin (Chang et al., 1980).
The degree of the recovery of activity is sensitive to a molar ratio of enzyme to
denaturant, time and pH of the incubation. Multidomains non-covalently associated in
proteins also exhibit disassociation and reassociation as entire units. While covalently
intact tit-thrombin refolds from urea in less than two minutes with complete return of both
clotting and esterase activity, three (noncovalent)-domain y-thrombin requires up to 90
minutes to regain full esterase activity under the same conditions (Chang et a1, 1980). It
was suggested that y—thrombin renaturation represents a two step process: (1) the rapid
refolding of the covalently intact units and (2) subsequent reassociation of the three non-
covalent domains of the protein to yield a lower energy, fully active conformation. A
possible side effect of the treatment of proteins with denaturating agents is self-
association or an oligomerization of protein molecules trapped in conformationally
altered forms (Zhuang et al., 1996”).

The main incentive to study the pattern of interactions between denaturants and
proteins at the molecular level is to gain insight into the nature of protein stability,
susceptibility to different levels and types of denaturants and structural rearrangements
associated with denaturation. It is expected that denaturating agents are not only
randomly distributed on the surface of a protein but also that they bind to specific
operating sites in characteristic patterns. A total of nine urea molecules have been
crystallographically identified in the structure of hen egg-white lysozyme from crystals

soaked in 9.0 M urea (Pike & Acharya, 1994). While eight of these nine molecules were

74

also found in lysozyme soaked in 4.0 and 5.0 M urea, only three urea binding sites were
occupied in the lysozyme structures of crystals soaked in 0.7, 2.0 and 3.0 M urea. Three-
dimensional X-ray intensities of crystals of (Jr-chymotrypsin dialyzed against either 2.0 M
guanidine hydrochloride or 3.0 M urea solutions at pH 3.6 were measured at 2.8 A
resolution and examined to identify denaturant binding sites (Hibbard & Tulinsky, 1978).
Guanidinium ions appear to have bound to the protein at two distinct locations on the
surface of the protein: (1) in the intermolecular uranyl heavy atom binding site (Tulinsky
et al., 1973; Vandlen & Tulinsky, 1973) and (2) in a solvent cavity in the dimer interface
region. Structural analysis of the urea treated crystal of chymotrypsin displayed changes
occurring not only on the surface of the protein but also in the hydrophobic interior close
to nonpolar side chains of Ile6, Val9 and Va123 of chymotrypsin. Surprisingly, neither
guanidine hydrochloride nor urea was found in the active site region of the protein
(Hibbard & Tulinsky, 1978).

Crystallographic identification and characterization of hydrogen bonding and van
der Waals interactions of denaturating agents bound to the active site region of an enzyme
could make significant contributions to the process of drug design. However, to present,
neither the sites by which proteins interact with denaturing agents nor the consequent
structural alternations upon binding of the denaturants have been classified or precisely
determined at the molecular level for most of the proteins. Factors, such as (1) poor
diffraction of protein crystals treated with denaturing agents and (2) conformational
heterogeneity of protein molecules upon treatment with denaturing agents have been

major obstacles to fiilly addressing the process of denaturation.

75

The multifunctional biological activity of thrombin resulting from the presence of
four operating sites, the active site, the fibrinogen and heparin binding exosite l and
exosite II and the sodium binding site (Chapter 2), stimulated our interest to study the
relationships between biochemical and structural alternations caused by denaturation to
this multifunctional enzyme. To fully address the problem: (1) all transition states from
the intact to fully denatured thrombin structure should be characterized; (2) the changes
that occur at the catalytic and the allosteric sites during denaturation have to be analyzed;
(3) the catalytic activity of thrombin must be measured as a function of the concentration
of the denaturant; and (4) observed and simulated structural changes have to be correlated
with the measured catalytic activity of the enzyme.

Due to fundamental limitations of crystallography, the technique can not address
all of the foregoing. Crystallography, nevertheless, may shed light on the onset of the
process of denaturation. At low concentrations of urea: (1) the thrombin-hirugen
complex should still be crystallizable and (2) the denaturating agent should not yet be
randomly localized on the surface but rather bound to specific sites on the protein.
Identification of urea binding sites in the active site region of thrombin could make a
significant contribution to the process of drug design and to an understanding of the

initial phase of protein denaturation.

II. EXPERIMENTAL
Crystallization: An approximately 7-fold molar excess of hirugen was added to a
fi'ozen sample of human a-thrombin solution (1.0 mg/mL in 0.75 M NaCl) at 4°C to form

a 1 :1 complex thrombin-hirugen complex. Bound to the fibrinogen exosite I of thrombin,

76

hirugen prevents autocleavage of the enzyme. In approximately 24 hours, the solution of
the binary complex was concentrated to about 7.0 mg/mL using a Centricon 10
concentrator with a molecular weight cutoff of 30 KDa in a refiigerated centrifuge at 4°
C. The hanging drop technique and repetitive macroseeding were applied to obtain and
enlarge the crystals, respectively, as well as to increase the concentration of urea in the
crystals to the point where the crystals stop growing and start to dissolve. All
crystallization procedures were carried out in a 4 uL drop consisting of 2 pL of the binary
thrombin-hirugen complex and 2 uL of a well solution at 4° C. First, thrombin-hirugen-
urea crystals were obtained in the drop over the well solution containing 30 % PEG 8000,
2.0 M urea in 0.1 M SPB, pH 7.3. Then, a crystal selected for X-ray diffraction was
enlarged to dimensions of 0.70 x 0.40 x 0.20 mm in a fresh 4 uL drop which was pre-
incubated for three days over a well solution containing 32 % PEG 8000 and 3.0 M urea
in 0.1 M SPB, pH 7.3. Finally, the crystal was transferred into a fresh drop pre-incubated
for four days over 36 "/0 PEG 8000 and 4.0 M urea in 0.1 M SPB, pH 7.3 and was left to
equilibrate for 12 days. The final concentration of urea in the thrombin-hirugen crystal
before X-ray data collection was between the initial concentration of the denaturant in the
drop (2.0 M) and the initial concentration of the denaturant in the well (4.0 M). Thus, the
final concentration of urea in the thrombin-hirugen crystal will be hereinafter defined as
(2.0+X) M and is expected to be between the (2.0-3.0) M concentration range.

Intensity Data Collection. The X-ray diffraction data from the thrombin-hirugen
complex co-crystallized in the presence of (2.0+X) M urea were collected with an R-

AXIS II imaging plate detector. The radiation generated from a Rigaku RU200 rotating

77

anode operating at 5 kW power with the fine focus filament (0.3 mm x 3.0 mm) was
monochromated (CuKa) and intensified by focusing with Molecular Structure
Corporation-Yale mirrors. The crystal to detector distance was 10.0 cm and the swing
angle was 10 degrees. The crystal scattered X-rays to 1.8 A resolution. Autoindexing and
processing of the measured intensity data were carried out with the Rigaku R-AXIS
software package (Higashi, 1990). Characteristics of the crystal and the intensity data
collection statistics are given in Table 4-1.

Preliminary structure analysis: Scaling the X-ray diffraction data between native
thrombin-hirugen and that crystallized in the presence of urea was employed to estimate
the degree of possible structural re-arrangements induced by the denaturant. Two
independent reflection data sets from two different thrombin-hirugen crystals (R-merge
3.1% and 3.5%) were merged to produce the data set that was chosen as native
(Vijayalakshmi et al., 1994). This native data set contains total 15,157 reflections with
R-merge of 6.8% (Table 4-1). The collected intensity data set of thrombin-hirugen
treated with urea contains 23,588 reflections with R-merge of 5.9 % (Table 4-1).
Comparison of the cell parameters between the native and derivative crystals indicates
shrinkage of the thrombin-hirugen-urea unit cells in all three directions; however, the
changes are marginal, all within two percent. The I/o(I) = 4 cut-off criteria was applied
to the native and derivative data sets to illuminate weak reflections. Then, the reflections
from two data sets were sorted, matched according to the Miller indices and sealed with

PROTEIN (Steigemann, 1974). The scale constant (k) and the average B-value between

78

Table 4-1: Crystal data and intensity data collection statistics of the native“ and urea-

treated thrombin-hirugen complex.

 

 

Native” Urea-treated
Space group C2c C2c
Cell constants (A):
a 71.75 70.59
b 72.39 71.23
c 73.52 72.42
[3 (deg) 101.3 100.5
Resolution (A) 2.3 1.8
Observations (I/o(I)>l .0) 36,665 36,134
outermost range (A) 4 2.5-2.3 2.5-1.8
I/o(I) (outermost range) 6.3 7.0
Independent reflections (I/o'(I)>1.0) 15,157 23,588
Redundancy 2.4 1 .5
Completeness (%) 83 71
outermost range (%) 70 56
R-merge (%) 6.8 5.9
outermost range (%) 9.7 8.5

 

a - Vijayalakshmi et al., 1994.
b - native data set consists of two merged independent data sets from two different
crystals.

c - four complexes per unit cell, one per asymmetric unit.

79

native (N) and derivative (D) data sets (IFD|2=k lFN|2 exp[-2(BD-BN)(sin0/7t)2] were 2.2 and
—3.1 A2, respectively. The preliminary crystallographic analysis was based on a total
2,880 pairs of equivalent reflections distributed into ten shells according to their mean
amplitudes (<F>) mean resolution ranges (<d>) and as a function of mean <F/o(F)>. The
merging R-values between the native and derivative data sets calculated for individual
shells ranged from 6.7% to 19.3% as a function of <F> (Table 4—2A), from 9.4% to
13.8% as a function of <d> (Table 4-2B) and from 9.6% to 15.8% as a function of
<F/o(F)> (Table 4-2C). The overall merging R-value between the native and derivative
data is 10.8%.

Structure Determination. The thrombin-hirugen—urea crystal belongs to space
group C2 with four ternary complexes per unit cell (one molecule per asymmetric unit).
The crystal structure was determined using thrombin coordinates of the isostructural
binary thrombin-hirugen complex (PDB code: 1HAH) (Vijayalakshmi et a1, 1994). The
initial coordinates were optimized to an R-value of 34.0% by a rigid-body refinement in
the (9.0-3.0) A resolution range using the X-PLOR program package (Brunger, 1992).
Additional positional refinement decreased the R-value to 26.7%. The structure of
thrombin optimized by X-PLOR was then refined with the program PROLSQ
(Hendrickson, 1985) and PROF FT (Finzel, 1987). After 3 cycles of overall and 6 cycles
of individual B-refinement, the R-value converged at 24.6% (7.0-2.5) A. At this stage,
hirugen and both intramolecular and intermolecular sodium ions, identical to those

initially described (Zhang & Tulinsky, 1997), were included based on the examination of

80

Table 4-2A: Rm -va1ues as a function of the structure factor magnitudes of the native

erge

and urea-treated crystals of thrombin-hirugen.

 

 

No. of Shell < F > Merging R-value
1 116.8 19.3
2 166.2 19.1
3 208.4 16.9
4 250.2 15.1
5 296.5 12.9
6 347.8 12.4
7 413.8 11.3
8 497.9 9.1
9 625.2 8.6
10 933.9 6.7

 

81

Table 4-2B: Rwy-values as a function of the resolution of the native and urea-treated

crystals of thrombin-hirugen.

 

 

No. of Shell < d > (A) Merging R-values
1 2.3 13.8
2 2.5 12.5
3 2.6 11.9
4 2.8 10.8
5 2.9 11.9
6 3.2 11.3
7 3.4 12.1
8 3.9 10.0
9 4.6 8 1
10 7.3 9.4

 

82

Table 4-2C: Rwy-values as a function of < F/o(F) > of the native and urea-treated

crystals of thrombin-hirugen.

 

 

No. of Shell < F/o(F) > Merging R-values
1 7.3 15.8
2 12.2 13.5
3 16.8 12.5
4 22.2 11.6
5 29.6 11.1
6 40.1 10.6
7 53.7 9.6
8 75.8 9.9
9 116.7 10.1
10 238.8 9.6

 

83

(2Fo—Fc) and (Fo-Fc) difference electron density maps. Extending the data further to 2.1
A, to 1.9 A and finally to 1.8 A and conducting further refinements resulted in converging
of the R-value to 23.1% (the model included thrombin, hirugen and two sodium ions; no
water or urea molecules). Thereafter, solvent and urea molecules were progressively
found and added into the refinement. The final structure contains five molecules of urea,
two sodium ions and 190 molecules of water with occupancies greater than 0.5. The final
R-value is 19.1 % (8.0-1.8) A. The final refinement and R-value statistics are

summarized in Table 4-3.

IV. RESULTS AND DISCUSSION

The presence of urea during the crystallization of the thrombin-hirugen complex
had little effect on the dimensions of the monoclinic unit cell (Table 4-1). The
denaturant, however, did have a substantial effect on crystal growth. To grow thrombin-
hirugen crystals suitable for X-ray diffraction in the presence of the denaturant, it is
necessary to increase the concentrations of both the protein complex and the precipitant
PEG and to lower the crystallization temperature to 4° C. It was noticed that the growth
rate of thrombin-hirugen-urea in the hanging drop over the well solution containing 30 %
PEG 8000, 2.0 M urea in 0.1 M SPB, pH 7.3 was significantly faster compared to that of
the native thrombin-hirugen. However, when the crystals were transferred into the fresh
pre-incubated drop over the well solution containing 32 % PEG 8000, 3.0 M urea and
then 36% PEG 8000 and 4.0 M urea, both in 0.1 M SPB, pH 7.3, the apparent crystal
growth rate became comparable to that of the native thrombin-hirugen in the case of

former and

84

Table 4-3: Refinement summary of deviations from ideality and final refinement

parameters of the thrombin-hirugen-urea complex.

 

 

Restraints Target RMSA

Distances (A):

Bond distance 0.015 0.011

Angle distance 0.020 0.021

Planar 1,4 distance 0.030 0.034

Planes 0.035 0.041
Chiral volume (A3) 0.15 0.18
Non-bonded contacts (A)

Single-torsion 0.60 0.20

Multiple torsion 0.60 0.25

Possible H-bond 0.60 0.24
Isotropic thermal factors (A2)

Main-chain bond 1.0 1.2

Main-chain angle 1.5 1.8

Side-chain bond 2.5 3.0

Side-chain angle 3.5 4.3

 

Resolution range (A)
No. of reflections
<B> (A2)
R-value (%)

8.0 — 1.8
21,529
24.1
19.1

 

85

then ceased. Attempts to further increase the concentration of urea in crystals resulted in
their dissolution.

Electron density was well defined for most of the residues of thrombin except the
termini of the A chain (residues ThrlH-AlalB, Ile14K-Arg15) and the autolysis loop
(residues Trp148-Lysl49E), which are typically disordered in other isomorphous
thrombin structures. A comparison of CA positions of thrombin in native thrombin-
hirugen (PDB code: 1HAH) (Vijayalakshmi et al., 1994) and refined thrombin-hirugen
co-crystallized in the presence of (2.0+X) M urea indicates no significant conformational
changes: the rmsA of CA positions is only 0.30 A. The refined thrombin-hirugen
structure is also similar to that of the PPACK- (PDB code: lPPB) (Bode et al., 1992) and
FPA- (PDB code: 1F PH) (Stubbs et al., 1992) thrombin bound complexes: rmsA are 0.33
A and 0.45 A, respectively. The conformational resistance of the main chain of thrombin
to urea denaturation at (2.0+X) M concentration of the denaturant is not surprising and is
consistent with the sigmoidal curve representing protein denaturation. The
conformational changes in thrombin become noticeable only at the inflection point of 3.5
M or higher concentrations of urea when protein unfolding occurs spontaneously within a
very narrow concentration range of the denaturant. All residues of the active site of
thrombin are essentially positioned optimally and remain so on urea binding in the
thrombin-hirugen—urea complex. The sidechain of Glul92 residue, a part of the active
site region of thrombin, adopts an extended conformation into solvent region similar to
those observed in the structures of Selectide and Molecumetics inhibitors as well as in the

native thrombin-hirugen (Vijayalakshmi et al., 1994). The “up” conformation of the

86

Glul92 side chain is stabilized by a water molecule. Furthermore, the Ramachandran
plot of the thrombin-hirugen-urea complex shows that 204 (83.3 %) residues of the total
245 non-glycine and non-proline amino acids of thrombin are in the most favorable
regions while 41 (16.7 %) residues occupy additionally allowed areas. There is no
residue in disallowed regions.

Five urea molecules have been clearly identified in the thrombin-hirugen complex
(Figure 4-1) based on the (2Fo-Fc) and Gio-Fc) difference electron density maps. Three
molecules of the denaturant (Urel-Ure3) occupy the active site region of thrombin and
can directly affect the catalytic activity of the enzyme. Two urea molecules (Ure4 and
Ure5) are located next to the heparin binding exosite II and may indirectly affect the
catalytic functions of thrombin by causing conformational changes at the active site of the
enzyme similar to those observed in heparin-ATIII inactivation of thrombin. It is also
known that changes at the fibrinogen exosite I or the sodium binding sites can have
allosteric consequences on both kinetic and thermodynamic characteristics of the enzyme
(Wells & DiCera, 1992). However, in the thrombin-hirugen complex, the fibrinogen
exosite I is occupied with hirugen that not only prevents autocleavage of the enzyme but
also occludes the site from being accessible to the urea molecules. Finally, based on the
examination of (2Fo-Fc) and (Fo-Fc) difference electron density maps, the two Na+
binding sites of thrombin-hirugen remain intact. To summarize, the primary binding sites

of urea in thrombin are in the active site region and in the heparin binding exosite of the

enzyme.

87

Hirugen
. ,‘ C—Terminal

  
 
 
  
  
   
 

Chain B
C—Terrninal

Hirugen
N-Terminal

C—Terminal

Figure 4-1: Urea binding sites of thrombin. Urel-Ure3 are in the active site region of the
enzyme. Ure4 and Ure5 are located adjacent to heparin binding exosite II. Ser195 of the

catalytic triad is numbered.

88

Three urea molecules and two water molecules (0,698 & 0,,700) adopt a
semicircular arrangement at the catalytic site stretching from the S1 to the S2 site (Figure
4-2). The first urea molecule (Urel) lies in the upper part of the specificity pocket, a
subsite that accommodates arginine-like residues. It is located approximately 3.3 A below
Ser195-OG and 5.5 A above Asp189-OD, at the position corresponding to CG-CD-NE of
Arg in thrombin-bound PPACK. This urea molecule is incorporated into an extensive
solvent network interacting with the enzyme via multiple hydrogen bonds and is
stabilized through two hydrogen bond bridges (0,668 and 0,695) that link Urel-N1 and
Gly219-O (Figure 4-3). In addition, two water molecules (0,661 and Ow668) mediate
hydrogen bonding between the urea molecule and the carboxylate oxygen atoms of
Asp189. The overall pattern of the interactions at the specificity site is essentially the
same as that found in crystal structures of thrombin-PPACK and many other thrombin-
inhibitor complexes in which an Arg residue occupies the P1 position.

The second urea molecule (Ure2) interacts directly with Ser195-OG of the
catalytic triad and with Gly193-N (2.71 A) and Ser195-N (3.14 A) of the oxyanion hole
of thrombin (Figure 4-3). It is positioned to mimic inhibitors with C-terminal carboxylate
or terminal peptide-like groups. Water molecule Ow698 adopts a distorted tetrahedral
arrangement coordinated by Ser195-OG, Ow683 and nitrogen atoms of Urel and Ure2
positioned almost edge to plane to each other (Figure 4-2). A third urea molecule (Ure3)
located in the active site region of thrombin utilizes dipol-rt electron mediated

interactions with Tyr60A (2.70 A) and Trp60D (3.70 A) at the P2 subsite similar to

89

 

Ser1 95

Asp189

Figure 4-2: Stereoview of the final (2Fo-Fc) electron density of Urel-Ure3 and their

water environment. Contoured at the lo level.

90

,Ow645

300 X279
szoo ------ N1

”‘32 ,. G1y193-N
. 2 71

N2 .0 '
; \245 ~~.‘3.14

2.57; 2.47 ‘~

I

. M’s 195 00 ‘Serl95-N
Ow683 -------- Ow698' 2.52 er '
2.58
2.50\
N2
Urel
2.50
Ow695 ----- {‘11 0‘
2.76”: x’ \‘ 272
x , 2.50 ‘.
G1y219-O . '2',§gb"o‘";°°§~.~2.62 0w504
3.005 0W5“
061 2.54
YODZ
Asp189-OD

Figure 4-3: Schematic diagram of hydrogen bonds (A) formed by Urel and Ure2 in the

active site region of the thrombin-hirugen complex.

91

cation-7t electron mediated interaction (Lin & Johnson, 1995). The n-faces of the
aromatic residues, Tyr60A and Trp60D are sufficiently rich in n-electrons to form not
only a hydrophobic pocket but also a cation-like recognition site. In addition, Ure3-O
forms hydrogen bonds with a water molecule 0,664 (3.00 A) and HisS7-ND1 (3.14 A) of
the catalytic triad of thrombin. The fourth and fifth urea molecules are located on two
different sides of the heparin binding exosite 11, approximately 5 to 7 A away from
residues Arg126, Arg165, Lysl69 and Arg233 and about 20 A apart from each other
(Figure 4-1). These two urea molecules form hydrogen bonds with the Leu130-O (2.98
A) and Ile147-O (3.00 A) of thrombin, respectively.

The two Na+ binding sites remain intact in the thrombin crystal structure upon
treatment with urea and are practically identical to those described previously (Zhang &
Tulinsky, 1997). Resistance to the effect of urea is most likely due to the extensive water
network around the sodium ions and octahedral coordination of Na+ by water molecules.
This results in the low mobility of the ions reflected in their occupancies, and temperature
factors (0 = 1.00, B = 20.6 A2, intramolecular Na“; Q = 1.00, B = 18.7 A2, intermolecular
Na”).

Even though thrombin shows no significant signs of denaturation in (2.0+X) M
urea, we have been able to crystallographically detect the primary urea binding sites in
thrombin. It comes as no surprise that the binding of the denaturant is selective: all
primary urea binding sites are localized at the operating functional sites of thrombin, such
as the active site and the heparin binding exosite 11. Identification and analysis of the

effect of urea on the crystal structure of thrombin-hirugen should be valuable to other

92

biochemical and crystallographic studies of blood proteins that are less resistant to
denaturation effects. It should also provide better insights into the molecular mechanism

of the denaturation process.

93

CHAPTER 5:

STRUCTURE-FUNCTION DETERMINANTS OF RECOMBINANT KRINGLE 5

OF HUMAN PLASMINOGEN

I. INTRODUCTION

Kringle domains found in non-catalytic regions in a variety of blood proteins
(Figure 1-7 & 1-8; Table 1-2) are autonomous structural and functional modular units of
approximately 80 amino acids cross-linked by three disulfide bridges in the pattern 1-6,
2-4 and 3-5 (Figure 1-9). Biological evidence suggests the ligand binding site (LigBS)
of most kringles binds lysine residues of several different proteins, such as fibrin, 0L2-
antiplasmin, tetranectin and thrombospondin, and is important in molecular recognition.
These kringle-dependent interactions can be competitively inhibited by lysine
zwitterionic analogues such as a-aminocaproic acid (EACA), 5-aminopentanoic acid (5-
APnA), 7-aminoheptanoic acid (7-AHpA), benzamidine, trans-4-aminomethylcyclo-
hexane-l-carboxylic acid (t-AMCHA) and p-amino-methylbenzoic acid (Table 5-1).
Three-dimensional structures of uncomplexed and liganded kringle domains of most of
the foregoing kringles have been determined by X-ray crystallography (Mathews et al.,
1996 and references therein) and by NMR methods (Rejante & Llinas, 1994 a’b).

Association of plasmin(ogen) with fibrin via the LigBS located in the K1 and K4
module essential for the digradation of the fibrin clot. The LigBS of plasminogen K1
(Wu et al., 1994; Mathews et al., 1996) and K4 (Mulichak et al., 1991; Wu et al., 1991) is

characterized by short arrays of residues His33-Arg35 (His33-Ly336 in K4), Asp55-

94

Asp57, Trp62-Phe64 (Tyr62-Tyr64 in K1) and Arg71-Tyr74, which together are arranged
in a relatively open elongated surface depression approximately 9 A wide and 12 A long.
Aromatic residues Trp62, Phe64, Trp72 and Tyr74 form a hydrophobic pocket bounded
by a negatively charged anionic subsite containing Asp55 and Asp57 at the top and a
positively charged cationic one containing Arg35 or Lys36 and Arg7l at the bottom. In a
display of polar efficiency, the anionic and cationic centers of the LigBS simultaneously
stabilize the amino and carboxylic group of ligands (Wu et al., 1991; Mathews et al.,
1996). The hydrophobic pocket interacts with the methylene chain of (o-amino acids so
that all three binding centers of the LigBS are simultaneously operative in an unrestricted

way.

Table 5-1: Kd (uM) values of ligand binding to various kringle modules.

 

 

Kringles 5-APnA EACA 7-AHpA t-AMCHA PnA HxA
Kl 24 12 250 1 N/A N/A

K4 29 26 280 5 3000 N/A

K5 580 140 367 22 168 470

K5 [L71R] 47 26 68 3 N/C N/C
KIV-10 66 20 230 N/A N/A N/A

 

 

 

PnA — 5 aminopentane; HxA - 6-aminohexane.
N/A — Dissociation constant is not available.

N/C - No fluorescent changes

 

Our interest in the structure of plasminogen K5 (Figure 5-1) lies in the fact that
this kringle module possesses a unique LigBS. Compared to the LigBS of K1 and K4,

the unique feature of the binding region of plasminogen K5 lacks a cationic center

95

 

 

 

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e 00 aeomooooeoo
p G
”“16 Wooomm 90009
e ., a .. o
0
0000 one...
u @0660

Figure 5-1: Primary structure of human plasminogen KS.

96

(Tables 5-2 & 5-3) so that a ligand to can only occupy the anionic binding site. In K5 the
residues positioned near the cationic center are His33-Ser34—Ile35 and Leu71. As a
result, the affinities of this domain for (tr-amino acids are weaker, but better for
alkylamines as compared to K1 and K4 (Table 5-1). In order to test the role of Leu71 in
the LigBS, the recombinant Leu71Arg mutant (K5[L71R]) was constructed, expressed
and purified (Chang et al., 1998). Using intrinsic fluorescence titration methods, the
dissociation constants of the wild type K5 domain as well as the K5[L71R] mutant have
been determined for various inhibitors. Comparison of the Kd values of K5 and
K5[L71R] for ligand binding clearly demonstrates the enhancement of the co-amino acids
for the mutant kringle (Table 5-1). The binding extent of K5 [L71R] for EACA is

essentially the same as that of K4 and only 2-fold weaker than the best affinity of K1.

II. EXPERIMENTAL

Crystallization: Previous experience with crystallization of kringles (Mulichak et
al., 1991; Wu et al., 1994; Mathews et al., 1996) suggested that PEG 400 to 8000 in Net"-
Hepes buffer, pH 7, with or without salt, would be more suitable for crystallizing K5. An
in-house set of factorial solutions utilizing Li2SO4 as salt (protein concentration of 60
mg/mL) produced small crystals using the vapor diffusion hanging drop method, from a
solution of 24% PEG 8000/0.15 M Li2S04 in 0.1 M Na+-Hepes, pH 7.0. The reservoir

contained 1 mL of the solution, and the hanging drop consisted of 1 uL of the protein

solution added to 1 uL of the reservoir solution. X-ray diffraction quality crystals with

97

  
  
  
    
  
  

 
 
 
 
 

 
 
 
 
 

  
  
  
  
  
   
   
  

 

   
    
 

1 5 10 15 20 25 30

K1 CKTGNGKNYRGTM KNIGTKTCOWSSSPHR
K2 CGMHCSENYGKI MSLEAOAGCQWESSPH
K3 CGGNYLKTERGNVAV VSGTCQWSPHHHAOTT
K4 CYHGGDOS SIT TTKKSMTGCOWSSPHR
K5 CMFGGNGK RAT VTGCTPQDWPHAAQE
KIV CYHGNGOS FIT VTRSMTGTCOWSSPHR

45 50 55 60 65 70 75 80
K1 HSEPGLEE NPDNEPQEGPWCYTTDPKRY CDILEC
K2 FNKNKKREL-RFNPLNYCRNPDPWCTTDPKR CDIPRC
K3 FPLECKND NPDGKPWR-AHTNSQV CKIPSC
K4 YPNGLATM NPDA K'GPWCFSVTTDPR CNLKKC
K5 NPGLERAK NPDG GVGPWCYTTmPKIYD CDVPQC
KIV YNDTPGLMNYCRNPDA T-PWCFTDMPSIWE CCNLTR

Table 5-2: Primary structures of plasminogen K1-K5 and apo(a) KIV-10 aligned in
respect of K1 to maximize conservation. The number of residues (1-80) standardized to
plasminogen K5. Deletion sites at positions 34 and 59 are indicated with -. Sequence
conservation is boxed: identical residues are in black boxes, homologous residues are in

the gray boxes.

98

1 5 10 15 20 25

 

 

human-K5 CMFGNGKGYRGKRATTVTGTPCQ WAA
bovine-K5 CME|GIIGKSYRGKATTVAGVPCQ
R
D

D

E
YRGKRATTVAGVPCQEWAA

G

Y R G K‘T‘AIVITIA A G T P C Q

30 35 40 45 50

WAA

 

porcine-K5 C M F G N G K
W A A

 

 

 

 

 

 

murine-K5 C MMG N G K

 

 

 

 

human-K5 QEPHRHSIFTPETNPRAGLEKNYCRNP

 

 

bovine-K5 QEPHEHSIFTPETNPQSGLEEINYCRNP
porcine-K5 QEPHRHSIFTPETNPRAGLEKNYCRNP

 

 

murine-KS QEPHRHSIFTPWTNPRAl—D—ILEKNYCRNP

 

 

 

55 60 65 7O 75 80

 

 

human-K5 DGDVGGPWCYTTNPRKLYDYCDVPQC

bovine-K5 DGDVNGPWCYTMNPRK‘DYCDVPQC
L

porcine-K5 DGDDNGPWCYTTNPQK DYCDVPQC

 

murine-KS DGDVNGPWCYTTNPRKLYDYCDIPLC

 

 

Table 5-3: Primary structures of the plasminogen K5 modules from the various species.

Identical residues are boxed.

99

 

dimensions of 0.3 x 0.15 x 0.05 mm were obtained by multiple macroseeding while the
PEG 8000 concentration was stepwise decreased fiom 24% to 22% to 18% and to 16%.

Intensity Data Collection: The X-ray diffraction data of the K5 crystals were
collected with an R—AXIS II imaging plate detector using Molecular Structure
Corporation-Yale focusing mirrors. The CuKor radiation was generated with a Rigaku
RU200 rotating anode operating at 5kW power with a fine focus filament (0.3 mm x 3.0
mm). Intensity data were measured at -150° C with crystals flash frozen in a cryosolvent
consisting of 20% PEG 4000, 20% glycerol, and 0.1M Na+-Hepes buffer, pH 7.0. The
crystal-detector distance was 10.0 cm and the detector-swing angle was 12 degrees.
Autoindexing and processing of the diffraction data were carried out with the Rigaku R-
AXIS software package (Higashi, 1990). The intensity data collection statistics are
summarized in Table 5-4.

Structure Determination: The crystal structure of K5 was determined by the
molecular replacement method using the coordinates of K1 (Wu et al., 1994) as an initial
model. All residues of K1 that differed in sequence from K5 were replaced with alanine
in the model and both N- and C-terminal interkringular peptides were ignored.
Rotation/translation searches were carried out with the program AMoRe (Navaza et al.,
1994) in the range 10.0-3.0 A resolution. The rotation search provided two separate
solutions with correlation coefficients of 0.28 and 0.19, corresponding to each of the two
molecules in the asymmetric unit related by a non-crystallographic 2-fold rotation axis
approximately parallel to the z-direction. The two solutions were also in agreement with
the self-rotation search. The position of one molecule of the rotated structure was

determined with a translation function that provided a R-value of 46.8% (correlation

100

Table 5-4: Crystal data and intensity data collection statistics of plasminogen K5.

 

Space group P21212

Cell constant (A)

a 77.43
b 79.20
c 30.78
Molecules/asymmetric unit 2
Solvent fraction (%) 42
Resolution (A) 1.66
Observations 42,366
Independent reflections 16,720
Redundancy 2.5
Outermost range (A) 1.80-1.66
R-merge (%) 4.2
outermost range (%) 8.7
Completeness (%) 72
outermost range (%) 37

1/0 outermost range 3.6

 

101

coefficient, 0.31); R was 50.1% (correlation coefficient 0.18) for the other molecule. The
coordinates a model of both rotated and translated molecules were optimized by rigid
body refinement to an R-value of 38.1% (correlation was 0.59). Additional positional
refinement was conducted with the X-PLOR program package (Brunger et al., 1992) by
energy minimization to relieve close contacts of the model. This decreased the R-value
to 33.5%.

Structure Refinement: The dimeric structure of K5 was refined with the program
PROLSQ (Hendrickson, 1985) and PROFFT (Finzel, 1987). The first stage of
refinement, (7.0 - 2.8) A, employed tight positional non-crystallographic symmetry
(NCS) and an overall B-factor. After three cycles, starting with an average B = 15 A2,
the R-value only changed from 35.7% to 34.5%. The next three cycles of the individual
B-refinement with tight NCS geometry and temperature factors restraints for atoms of
both the main and side chains (0.5 A, 3.0 A2), and six cycles of looser NCS geometry
and temperature factors restraints (1.0 A, 10.0 A2) converged at R = 31.9%. Continued
refinement using alternate tight-loose NCS restraints decreased R-value to 26.5%. At this
stage, the (2Fo-Fc) electron and (Fo-Fc) difference density maps had most of the side
chains of K5 that were arbitrary alanine residues in the initial model. The insertion
residue (Ser34) with respect to K1 (Table 5-2) was fixed based on an analysis of the
electron and difference density maps after deletion and then reconstruction of the His33-
Thr37 region. This clearly showed that the insertion was Ser34. Further refinement was
conducted including most of the K5 residues in the model. Solvent water molecules was
found and added periodically, by examining difference density maps, during the

refinement at 2.5 A and higher resolution. In the final stage, beyond 2.1 A resolution,

102

the NCS restraints were abandoned and the two molecules of K5 were refined
independently. Summary of final refinement parameters and their deviations are listed in

Table 5-5.

III. RESULTS

The X-ray crystal structure of K5 has been employed to examine critical aspects
of the LigBS of this kringle as it relates to those of K1 and K4. Crystals of K5 were
grown in the orthorhombic space group P21212 with dimensions 77.43 A x 79.20 A x
30.78 A (Table 5-4). The asymmetric unit contains two molecules (Figure 5-2) related by
a non-crystallographic 2-fold rotation axis approximately parallel to the z-direction. The
final model converged at an R-value of 16.6% at 1.66 A resolution and consisted of 1294
protein atoms and 193 water molecules with occupancy > 0.6. Two K5 molecules in the
dimer are almost identical, which especially applies to the LigBS regions (Figure 5-3).
The rmsA between the superimposed CA atoms of the two K5 units is 0.31 A. The
Ramachandran plot of the two kringles, generated by PROCHECK (Laskowsky et al.,
1993) shows that 106 (81.5%) residues of a total of 130 non-glycine and non-proline
amino acids occupy the most favorable regions, while 22 (16.9%) residues are in
additionally allowed areas (Figure 5-4). Similar to other kringle structures, such as K1
(Mathews et al., 1996), K4 (Mulichak et al., 1991), KIV-10 (Mikol et al., 1996;
Mochalkin et al., 1999), the residue Lys48, which is located in a hairpin turn, is in a
generously allowed area of the Ramachandran plots. Notwithstanding, it has well-defined
main and side chain electron density. Moreover, the Ramachandran plot of K5 indicates

a close coupling of the (a, u/angles of the two molecules in a K5 dimer (Figure 5-4).

103

Table 5-5: Refinement summary of deviations from ideality and individual

refinement statistics of plasminogen K5.

 

 

 

Target RMSA

Distances (A):

Bond lengths 0.020 0.016

Bond angles 0.040 0.034

Planar 1-4 0.050 0.050
Planes (A):

Peptides 0.030 0.032

Aromatic groups 0.030 0.030
Chiral volumes (A3) 0.130 0.160
Non-bonded contacts (A)

Single torsion 0.60 0.18

Multiple torsion 0.60 0.24

Possible H-bond 0.60 0.23
Thermal parameters (A2)

Main-chain bond 1.5 1.4

Main-chain angle 2.0 1.7

Side-chain bond 2.5 3.5

Side-chain angle 3.0 4.1
Refinement range (A) 9.0-1.66
Final R—value (%) 16.6

<B> (A2) 15.4

PDB code SHPG

 

104

 

 

Figure 5-2: Space filling and ribbon representations of plarninogen K5 dimer viewed

approximately down the local 2-fold axis in the crystal structure.

105

 

Figure 5-3: Superposition of the LigBS of K5 molecules 1 and 2 of the dimer. A residue

of each segment is labeled. Molecule 1 is shown in gray. Molecule 2 is colored black.

106

 

Ramachandran Plot
Plasrninogen K5

 

Psi (degrees)

 

 

Phi (degrees)
Plot statistics

Residues In most favoured regions [A3, L1 1% 111 .5'}

addil rum] allowed regions [n.bJ pl 22 17.0%
Residues m gcncnvuxly allowed regions l-a. -h. -1 -pl 2 1.5%
Residues in disallowed regio m 0 .0?
Number of non-glycine and nun-proline residues 130 100.0%
Number of cercsrdues (excl Gly and Pro) 2
Number of glycine residues (shown as unngles) 18
Number 01 proll ne residues 1!
Total number 01 rcsrducs 168

.411.

L n.
In have over 90‘} In the ml l'nwnled region

 

 

 

Figure 5-4: The Ramachandran plot of the final structure of the K5 dimer. Lys48

residues of molecule 1 (Lysl48) and molecule 2 (Ly8248) are in a generously allowed
region. Glycine residues are shown as triangles. Non-glycine residues are shown as

squared boxes.

107

The interface between the 2-fold rotation-related kringles consists of an antiparallel
arrangement of the Leu7l-Pr083 segments of the C-terminus of each kringle molecule.
Of the 34 water molecules hydrogen bonding to these two segments, seven hydrogen
bond with both, mediating the dimeric interaction between the two K5 molecules. Direct
hydrogen-bonding interactions also occur between these regions in a 2-fold manner, near
their ends, and involve Lys70, Asp73, and Ala82. They also occur close to the 2-fold
axis involving Asp76, while seven hydrophobic residues from each molecule participate

in stabilizing non-polar contacts.

IV. DISCUSSION

Kringles have a propensity to naturally occur in tandem arrays and associate with
one another to form more compact units (Mangel et al., 1990; Ramakrishnan et al., 1991).
The latter consideration also applies to crystal structures when more than one kringle
molecule constitutes the asymmetric unit (Padmanabhan et al., 1994; Mathews et al.,
1996). Although a number of different arrangements utilizing local rather than
crystallographic symmetry elements have been identified in crystal structures, such as 2-
fold rotation axis and pseudo 21 and 31 screw axis-like symmetries, none corresponds to
the unique antiparallel C-terminal interface arrangement of K5 in which 389 A2 or 13%
of kringle surface area is involved in the formation of the dimer (Figure 5-2). However,
the most distinguishing characteristic of the K5 dimer, as compared to those of other
kringles, is the large number of water molecules mediating the dimer interface. Whether
this or any of the other known kringle associations in crystals are physiologically relevant

remains to be shown by structure determination of a tandem kringle array.

108

Similar to other kringle modules, the LigBS of K5 is defined by His33-Ile35,
Asp55-Asp57, Trp62-Tyr64 and Leu71-Tyr74 as an elongated depression on the kringle
surface that is lined with solvent molecules (Figure 5-5). The hydrogen bonds that occur
in the LigBS of K5 are provided in Table 5-6. Those listed from Asn53 to Thr65,
especially when involving water molecules, reflect the excellent 2-fold symmetry
between two independent K5 molecules. The 16 hydrogen bonds in each LigBS strongly
support the notion that the LigBS of kringles is essentially preformed prior to, and
independent of, ligand binding. In the case of molecule 1, residues Gln28, Arg32 and
Ser34 from molecule 2 of the neighboring dimer make several hydrogen bonds that do
not possess 2-fold symmetry because of local rather than crystallographic symmetry
relationships between these contacts. The Arg32 of the neighboring molecule limits
access to the LigBS of molecule 1 in the crystal (Figure 5-5). However, in molecule 2,
the LigBS is essentially unobstructed. It is also noteworthy that the LigBS of molecule 1
is systematically occupied by eight water molecules (Figure 5-5), while molecule 2,
which lacks intermolecular contacts in this location, contains only five water molecules.
In fact, the (2Fo-Fc) electron density of seven of the water molecules of molecule 1 is
connected at the 10 level, resembling that of ligand binding. This apparent relationship
to ligand binding is probably the result of the intermolecular interaction of the LigBS
with Gln28, Arg32 and Ser34 from the adjacent molecule(s), which decreases the
mobility of the water molecules at the LigBS of K5.

In plasminogen K1, K4 and apo(a) KIV-10, residue Asp55 of the LigBS is in the

same orientation in both inhibited and unliganded kringles (x; z 60°). 1n plasnrinogen

109

 

Figure 5-5: Specific residues of K5 potentially present in the LigBS of molecule 1 of the
K5 dimer. The regions of the kringle represented are His33-Phe36, Asp55-Asp57,
Trp62-Tyr64, Leu71-Tyr74 are shown in gray. A residue of each segment is labeled.
Water molecules common to the LigBS of both kringles of the dimer are 1, 4, 5, 7 and 8;
those in molecule 1 only are 2, 3 and 6. 1=Ow524, 2=Ow525, 3=Ow419, 4=Ow565,

5=Ow589, 6=Ow524, 7=Ow526, 8=Ow527 of the PDB coordinate file (PDB code: SHPG).

110

 

Table 5-6: Hydrogen bonds (A) in the LigBS region of K5

 

 

Molecule 1 Molecule 2
Asn53-OD1 Asp57-N 2.84 2.83
-OD1 Ow540/470 2.93 2.97
-ND2 Asn57-O 2.70 2.70
-ND2 Asp57-O 2.97 2.94
Asp57-OD1 Gly59-N 2.81 2.93
-OD2 Gly60-O 3.12 2.95
Pro61-O Cys75-N 3.02 2.99
Trp62-N Arg52-O 2.91 2.87
-O -N 2.94 2.98
-NE1 Ow540/470 3.10 2.92
Cys63-N Asp73-O 2.87 2.96
-O -N 2.93 2.98
Tyr64-N Ow502/41 8 2.97 2.84
-O Gln23-N 2.61 2.81
-0H Ow526/425 2.71 2.84
Thr65-N Lys71-O 2.97 2.92
Tyr72-N Ser34-0Ga 2.90
-O Ser34-0Gan 2.55
-O Ow582a 2.76
-0H Ow491° 2.64
-0H Gln28-OE1° 2.81
Tyr74-OH Arg32-NH2a 3.00

 

 

a Neighboring molecules in crystal; hydrogen bond has no local 2-fold rotation

equivalent.

111

K5, however, Asp55 extends into the solvent region in a different orientation (X1 z 100°)
apparently nonconducive to ligand binding (Figures 5-3, 5-5 & 5-6). It is most likely that
the Asp55 residue of K5 reorients on ligand binding to form a doubly charged anionic
center along with Asp57, which then interacts with the amino group of ligand as it is
observed in the K1 and K4 (Figure 5-6).

When structurally superposed on K1, the segment His33-Thr37 of K5 contains
residue Ser34, which is deleted in Kl. This part of the kringle molecule appears to
possess the most flexibility (de Vos et al., 1991; Ami et al., 1993) and represents the only
region of the backbones of a number of kringle domains that differ from each other.
Although the difference between CA atoms of Ser34 of the two K5 molecules is 1.1 A,
the main chain and side chains of H1533 and Phe36 compensate for this displacement of
Ser34 and adopt the same orientation in both molecules (Figure 5-3). This is probably
due to the hydrophobic edge-face interaction of Phe36 with Tyr64 and Trp62,
respectively.

On the basis of similarities with K1 (Menhart et al., 1991; Mathews et al., 1996),
and K4 (Wu et al., 1991; McCance et al., 1994), the cationic residues of K5, Arg32,
Arg69 and Lys70, are candidates for interactions with the carboxylate of the ligand.
However, as highlighted in Figures (5-5) these residues are either clearly removed from
the binding pocket of K5 and/or have unfavorable orientations for interaction with the
ligand. This is also revealed by the distances of the functional groups from side chains of
Asp55, Asp57, Trp62 and Tyr72 residues that are uniformly critically positioned in the
LigBS in the other ligand binding kringles. Examination of all other cationic side chains

of K5 shows that none can contribute to the putative ligand pocket,

112

 

Figure 5-6: Superposition of the LigBS of K1 and K5 of plasminogen. K5 is in gray; K1
is shown in black. K1 is inhibited with EACA in the EACA binding pocket. All residues

are numbered according to K5.

113

except
0. ln 0
moiety.

180°.

was cc
Using
ApnA
first ft
A\-1C I
obser
the $2
the h;
nafur
h311rt
Wild
belyr
for

n”tor

llga

except possibly His33 (Figure 5-6). However, His33-NE is hydrogen bonded to Trp25-
O. In order for this group to play a role as a cationic donor to the ligand carboxylate
moiety, the orientation of the imidazole would have to be repositioned by approximately
180°.
To test the importance of Leu71 in the LigBS, the recombinant mutant K5 [L71R]
was constructed, expressed in the same yeast system, and purified (Chang et al., 1998).
Using intrinsic fluorescence titrations, the binding (dissociation) constants of K5 for 5-
ApnA, EACA, 7-AhpA, t-AMCHA, PnA and HxA have been determined along with the
first four for K5[L71R] (Table 5-1). The magnitudes of the latter, and in the case of t-
AMCHA, the sign, of the fluorescence changes possess some differences to those
observed for K5. This indicates that the environment of the residues, likely Trp62, is not
the same in two ligand-bound kringles. Such dissimilarities could include the nature of
the hydration of the LigBS, the orientation of the ligand in the binding pocket, and/or the
nature of side-chain-reporter group contacts. Since the ligand must bind in the more
hydrophilic pocket of the mutant quite differently than in the more hydrophobic site of its
wild-type counterpart, the differences in the intrinsic fluorescence of the ligand-saturated
polypeptide are not surprising. In any case, this intrinsic fluorescence change is saturable
for all of the ligands that exhibit binding, and, thus can be effectively employed to
monitor ligand-kringle interactions (Chang et al., 1998). The Kd values obtained for the

ligand-K5 [L71 R] interactions are also compared to those for other kringle domains.

114

and B
via ti
has t

(Scar

eXpr
mor
the

aln
rOle

C01-

Li.
no

hu

CHAPTER 6:

RECOMBINANT KRINGLE KIV-10 MODULES OF HUMAN
APOLIPOPROTEIN (a): STRUCTURE, LIGAND BINDING MODES AND

BIOLOGICAL RELEVANCE

I. INTRODUCTION

Plasrninogen is known to bind lysine-Sepharose via the LigBS located in the K1
and K4 modules (Miles & Plow, 1990) while Lp(a) (Figure 1-8) binds lysine-Sepharose
via the LigBS located in apo(a) KIV-10 (Figure 1-9; Table 6-1) (Eaton et al., 1987). This
has been supported by studies of defective lysine binding of intact rhesus monkey Lp(a)
(Scanu et al., 1993) and human Lp(a), both with Arg72Trp mutation in KIV-10 (Scanu et
al., 1994), as well as wild type KIV-10 (Trp72) and mutant KIV-10 (Arg72) individually
expressed in E. Coli (LoGrasso et al., 1994; Klezovitch & Scanu, 1996). Apo(a) of rhesus
monkey differed from the human counterpart by having: 1) an unpaired catalytic triad in
the protease region, 2) Arg in position 72 in the LigBS of KIV-10 and 3) no copy of KV,
a unit located between KIV-10 and the catalytic domain of human apo(a). The critical
role of Trp72 in lysine binding, which has emerged fi'om the above observations, is
consistent with the results of the structure of PT-Kl (Tulinsky et al., 1988), which has an
Arg in position 72. This residue extends diagonally across the region equivalent to the
LigBS, occupying it and sterically precluding binding of lysine-like ligands. The same
notion also emerges from the modeling of the LigBS of rhesus (Scanu et al., 1993) and

human mutant KIV-10/W72R (Scanu & Edelstein, 1995).

115

KNJO
K1

K3

K4

K5
PToKi
PT-KZ
l-PA K1
t-PA K2
u-PA K'

KlV-10
K1

K4

PT-K1
PT~K2
tPAi
t-PA 1'
u-PA

C0:

tht

KIV-10
K1

K3

K4

K5
PT-K1
PT-K2
t-PA K1
l-PA K2
u-PA K1

KIV-10
K1

K3
K4

PT-K1
PT-K2
l-PA K1
t-PA K2
u-PA K1

 

 

 
   
   
 
 

HGNGQS TCOSWSSM

GNGKN TCQKWSS
GEN

G GEN

HG GOS

YTAQNPSA
YHAHRSDA

PREK-YDYCDILE
SQV-WYCKIPS

PYCRK-LYDDVPQ
PETL-HRECSVPV
OPG-DFEYCIDNY
AGKYSSEICSTPA
NRRLTVVYDVPS
GLKPLVQECMVHD

Table 6—1: Primary structures of various kringles aligned in respect of apo(a) KIV-10 to

maximize conservation. The number of residues (1-80) standardized to plasminogen K5.

Deletion sites at positions 34, 42A, 47A, 59 and 70A are indicated with -. Sequence

conservation is boxed: identical residues are in black boxes, homologous residues are in

the gray boxes.

116

regi01
with

from

2) on
Howe
functi
KIV-1
KIV-1
Scanu

deficit

et al.,
Trp62
elong;
earliet
hydro
and A
at the
Ling
1991;
of (m

Opera

Recently, Chenivasse et al., (Chenivasse et al., 1998) reported that the C-terminal
region of apo(a) of chimpanzee (Pan troglodytes) exhibits a high degree of homology
with the corresponding region of human apo(a). In turn, chimpanzee apo(a) differed
from the rhesus counterpart by having: 1) an intact catalytic triad in the protease region,
2) one copy of the KV motif and 3) Trp in position 72 in the LigBS of KIV-10.
However, like the rhesus product, chimpanzee Lp(a) was deficient in lysine binding, a
functional impairment attributed to the presence of Asn instead of Asp at position 57 in
KIV-10 (Chenivasse et al., 1998). Another sequence polymorphism, Met66Thr, in apo(a)
KIV-10 has also been reported in 42-50% of the human population (Kraft et al., 1995;
Scanu et al., 1995). This mutation, however, is silent and causes no lysine binding
deficiency in Lp(a).

Like K1 and K4 of human plasminogen, the LigBS of apo(a) KIV-10 (Mochalkin
et al., 1999) is characterized by short arrays of residues His33-Arg36, Asp55-Asp57,
Trp62-Phe64 and Arg71-Tyr74, which together are arranged in a relatively open
elongated surface depression of approximately 9 A wide and 12 A long. As mentioned
earlier (Chapter 5), aromatic residues Trp62, Phe64, Trp72 and Tyr74 form a
hydrophobic pocket bounded by a negatively charged anionic sub-site containing Asp55
and Asp57 at the top and the positively charged cationic one containing Arg36 and Arg71
at the bottom. In a display of polar efficiency, the anionic and cationic centers of the
LigBS simultaneously stabilize the amino and carboxylic group of ligands (Wu et al.,
1991; Mathews et al., 1996). The hydrophobic pocket interacts with the methylene chain
of (ti-amino acids so that all three binding centers of the LigBS are simultaneously

operative in an unrestricted way. This binding mode will be hereinafter called embedded.

117

L _.___..2 ..__-.

observ
et al.. '
t0 intei
extend
leaving
be her
might
Arg711
thus f0
larger?
(Mikol
SUgges
A11 th
(Kd=2
(Table
differ
intera.
with .
LOGI'
anom
LigB

Struc

An unexpected binding mode in KIV-IO/Thr66 complexed with EACA was
observed by the crystallography group at Sandoz Pharma AG, Basel Switzerland (Mikol
et al., 1996). In this case, only the e-amino group of the zwitterionic ligand was reported
to interact with the anionic binding center of the kringle while the remainder of the ligand
extended into the solvent region, almost perpendicular to the surface of the kringle,
leaving the cationic site and the hydrophobic pocket unoccupied. This binding mode will
be hereinafter called unbounded, and such a structure (Sandoz KIV-10/Thr66/EACA)
might be expected with EACA bound to plasminogen K5 where the Arg3511e and
Arg7lLeu substitutions in the LigBS remove the cationic site of the kringle and could
thus force a ligand to solely interact with the anionic binding site with a correspondingly
larger K, (140 uM) (Table 5-1). The unbounded binding mode of KIV-10/Thr66/EACA
(Mikol et al., 1996), however, is not consistent with biochemical studies, which clearly
suggest the embedded binding mode displayed by K1 and K4 (Mochalkin et al., 1999).
All three binding centers are present in KlV-lO/Thr66 and the Kd value of EACA
(Kd=20 uM) is similar to those of plasminogen K1 (Kd=12 11M) and K4 (Kd=26 11M)
(Table 5-1). Futherrnore, the Kd values of ligands in the unbounded mode should not
differ significantly with the length of the ligand because only the terminal e-amino group
interacts with the anionic center. The measured dissociation constants of KIV-10/Thr66
with 5-APa (Kd=66 uM), EACA (Kd=20 uM) and 7-AHa (Kd= 230 11M) (Table 5-1;
LoGrasso et al., 1994) suggest otherwise and the embedded binding mode. These
anomalies and inconsistencies led us to investigate the binding mode of EACA in the
LigBS of human K1V-10/Thr66 during the course of a more extensive study of the

structures of the KIV-lO/M66 variant in its free and ligand-bound states.

118

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II. EXPERIMENTAL

Crystallization. Relatively large crystals of the KIV-10 variants, with and without
EACA, could be grown from ammonium sulfate-PEG 8000 solutions. Their X-ray
diffraction patterns, however, showed a complex splitting pattern that could not be
successfully interpreted and indexed. All indications suggested that the apparently single
crystals were actually several small, similarly oriented fused crystals. Consequently,
hanging drops of Hampton factorial solutions were examined. Excellent X-ray quality
single crystals of KIV-10/Met66/EACA and KIV-10fI’hr66/EACA could be readily
grown from Hampton condition #40 (Table 6-2). However, crystallization of the other
two kringles was not successful with this condition. Previous experience with kringles
suggested PEG 8000 as precipitant in a buffer with pH around 7.0, with or without salt,
might be suitable. An in-house set of factorial solutions based on the foregoing was
prepared and the hanging drop method applied again to the KIV-IO/Met66 and KIV-
10/W72R samples. The conditions were eventually refined to produce X-ray quality
single crystals. The full crystallization conditions and crystal dimensions of all the KIV-
10 kringles are given in Table 6-2.

Intensity Data Collection. The X-ray diffraction data of the KIV-10 crystals were
measured with an R-AXIS II imaging plate detector using Molecular Structure
Corporation-Yale focusing mirrors. The CuKor radiation was generated with a Rigaku
RU200 rotating anode Operating at 5kW power with a fine focus filament (0.3 mm x 3.0
mm). Intensity data were measured at -150° C with crystals flash frozen in a cryosolvent
consisting of 20% PEG 4000/8000, 20% glycerol, and 0.1M buffer that was also used in

the crystallization (Table 6-2). The crystal-detector distance was 10.0 cm

119

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120

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and the detector-swing angles were zero degrees for KlV-lO/Met66 and KIV-10/W72R,
eight degrees for KIV-10/Met66/EACA and ten degrees for KIV-IO/Thr66/EACA.
Autoindexing and processing of the diffraction data were carried out with the Rigaku R-
AXIS software package (Higashi, 1990) and the results are summarized in Table 6-3.
Three of the crystals are isomorphous; the c-axis of the KIV-10/W 72R mutant, however,
shrank by about 7.0 A.

Structure Determination. The crystal structure of KlV-lO/I'hr66/EACA was
determined by molecular replacement using the coordinates of K4 of human
plasminogen (Mulichak et al., 1991) (PDB code: 1PK4) as an initial model. All residues
of K4 that differed in sequence from KIV-10f1" hr66 were replaced with alanine.
Rotation/translation searches were conducted with the program AMoRe (Navaza, 1994)
in the resolution range (10.0 - 3.5) A. The rotation search provided one distinct solution
with a correlation coefficient of 0.30. The position of the molecule was fixed by a
translation function that increased the correlation coefficient to 0.39 (R=48.5%). Rigid
body optimization further increased the correlation coefficient to 0.64 (R=37.6%).
Additional positional refinement by energy minimization in the (10.0 - 3.0) A range to
relieve close contacts of the model was carried out with the X-PLOR program package
(Brunger, 1992) decreasing the R-value to 27.6 %.

The crystal structure of KlV-lO/Thr66 complexed with EACA was refined with
the programs PROLSQ (Hendrickson, 1985) and PROF FT (Finzel, 1987). First, three
cycles of overall B-factor refinement starting with B = 18.0 A2 converged to an R-value

at 32.9% (7.0 - 2.8) A. Then three cycles of the individual temperature factor refinement

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122

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brought the R-value to 29.4%. Further refinement applying alternate tight-loose

geometry restraints decreased R to 28.3%. At this stage, (2Fo-Fc) electron and (Fo-Fc)
difference density maps indicated most of the correct side-chains of KIV-lO/Thr66 that
were alanine residues in the initial model and revealed EACA, which was not included in
calculations. Further refinement was conducted with most of the actual residues of the
kringle, as well as EACA. Solvent water molecules were added periodically based on
examination of difference density maps at 2.5 A and higher resolution.

Since the crystals of KIV-IO/Met66 and KIV-10/Met66/EACA are isomorphous
with that of KIV-10/Thr66, the structure determinations of the first two were conducted
using isomorphous replacement methods. The refined coordinates of the KIV-lOfl'hr66
variant, with Thr66 replaced by alanine, were used as an initial model. Rigid-body
optimization with the X-PLOR program package in the (10.0 - 3.0) A range decreased
the R-value from 38.6 % to 33.2 % for unliganded KIV-IO/Met66 and from 43.7 % to
34.5 % for KIV-IO/Met66/EACA (without the ligand in the calculations). Additional
positional refinement improved the R—values to 25.1% and 28.1%, respectively. After
three cycles of overall B-factor refinement and three cycles of individual B refinement
using program PROFF T, R converged to 24.6% for KIV-lO/Met66 and 26.4% for KIV-
10/Met66/EACA. The (2F o-F c) electron and (Fo-Fc) difference density maps clearly
showed Met66 in KIV/Met66 and the EACA in addition, in the KIV-lO/Met66/EACA
structure.

The crystal structure of KIV-lO/W72R had to be determined de novo by the
molecular replacement method (because of the dramatic shrinkage of the c-axis, Table 6-

3) using the coordinates of KIV-10 from the KIV-10/Thr66/EACA complex. The

123

residu
model.
one dis
further
square

Table 1

variant
SU'UCtL‘
indica‘
Ramat
residu
PlaSm
1998)

48 ha

residues Thr66 and Arg72 were replaced with alanine and EACA was omitted from the
model. The rotation/translation search in the resolution range (10.0 - 3.0) A provided
one distinct solution with a correlation coefficient of 0.71 and a R-value of 32.6%, while
further rigid-body refinement decreased the R-factor to 30.5%. Final restrained least-
squares parameters and R-factor statistics for the KIV-10 complexes are presented in

Table 6-4.

[11. RESULTS

The electron density is well defined for most of the residues of the kringle
variants except the interkringle peptides that are disordered here and in other kringle
structures. The rmsA of the position of CA atoms between the different KIV-10 units
indicates that the molecules exhibit very similar structural folding (Table 6-4). The
Ramachandran plots of the KIV-10 variants reveal that all non-glycine and non-proline
residues, except Met48, lie in the allowed regions. Similar to other kringle structures,
plasminogen K1 (Mathews et al., 1996), K4 (Mulichak et al., 1991), K5 (Chang et al.,
1998) and unbounded KIV-lO/Thr66/EACA (Mikol et al., 1996;), the residue at position
48 has well-defined main and side chain electron density in all of them corresponding to
a hairpin turn but is in a generously allowed area of the Ramachandran plot. No kringle
residue was found in a disallowed region. As in other kringle structures, the Pro30

residue is in a cis conformation.
The LigBS of KIV-10 is defined by His33-Arg36, Asp55-Asp57, Trp62-Phe64, and
Arg71-Tyr74 (Figure 6-1). Hydrogen bonds in the LigBS region can be divided into

three principal groups according to several factors, which can depend on the ligand

Table 6-4: Summary of final restrained least-squares statistics of KIV-10 modules.

 

KIV- l 0/ KIV-1 0/ KIV-1 0/ KIV- l O/
Met66 Met66/ Thr66/ W72R
EACA EACA

 

Target RMSA RMSA RMSA RMSA
Distances (A):
Bond lengths 0.020 0.012 0.017 0.016 0.011
Bond angles 0.040 0.032 0.039 0.039 0.032
Planar 1-4 0.050 0.043 0.047 0.047 0.041
Planes (A):
Peptides 0.030 0.024 0.029 0.027 0.026
Aromatic groups 0.030 0.029 0.037 0.034 0.029
Chiral volumes (A3) 0.130 0.125 0.165 0.169 0.118
Non-bonded contacts (A):
Single torsion 0.60 0.18 0.18 0.20 0.22
Multiple torsion 0.60 0.26 0.21 0.28 0.29
Possible H-bond 0.60 0.32 0.24 0.32 0.28
Thermal parameters (A2):
Main-chain bond 1.5 1.1 1.5 1.6 1.1
Main-chain angle 2.0 1.8 1.8 2.2 1.7
Side-chain bond 2.5 2.1 2.6 3.0 1.8
Side-chain angle 3.0 3 .0 3.3 3.5 2.5
Range (A) 9.0-2.1 9.0-1.8 9.0-1.8 9.0-2.2
R-value (%) 17.8 17.6 18.2 16.6
No. of water molecules8 67 77 109 41
<B>(A2) 21.8 15.6 15.9 21.5
CA RMSA (21)" 0 0.30 0.30 0.43
PDB code IKIV 2KIV 3KIV 4KIV

a - Occupancy > 0.5
b- Calculated versus KIV-10/Met66

 

125

 

Figure 6-1: Superposition of EACA of different lysine binding kringles. KIV-
10/I‘hr66/EACA side chains of LigBS are in gray. Embedded binding modes are labeled:
EACA of K1 (1); EACA of K4 (2), EACA of KIV-10/Thr66/EACA (3). EACA of KIV-

10/Thr66/EACA (Sandoz) displays the unbound binding mode. Note different

conformations of Arg36 in KIV-10/Thr66/EACA and in KIV-10/Met66.

126

(EACA) or on intermolecular interactions of neighboring molecules in crystals. The
first group includes hydrogen bonds that are common to all the structures (Table 6-5).
These interactions in the LigBS mainly involve atoms of the backbone and are generally
not completely exposed on surface. The second group consists of the hydrogen bonds
that result from the binding of EAC A ligand (Table 6-6). The residues of the anionic and
cationic binding centers and several water molecules in the LigBS participate in the
hydrogen bonding interactions of this group. Residue Arg32, which is located on the
surface of the LigBS, gives rise to the third group of hydrogen bonds in the LigBS region
that depend on crystal packing.

In the liganded structures of the Met66 and Thr66 variants of KIV-10, the electron
density of the EACA in the LigBS is similar and well defined (Figure 6-2) and EACA
interacts in the embedded way with all three binding centers in both kringles (Figures 6-1
& 6-2). The anionic (Asp55/Asp57) and cationic (Arg36/Arg71) centers stabilize the
amino and carboxylic groups of EACA, respectively, while the hydrophobic pocket
(Trp62, Phe64, Trp72, Tyr74) interacts with the methylene chain of the ligand. Thus, the
KIV-IO/Thr66/EACA structure determined here differs very significantly from the
unbounded KIV-10/Thr66/EACA structure reported previously (Mikol et al., 1996),
which only showed ligand binding at the anionic center (Figure 6-1). Except for Arg36,
the binding of EACA to both the Met66 and Thr66 variants occurs with minimal
structural reorganization so the LigBS appears to be essentially preformed anticipating
ligand.

Because of the decrease of the lysine binding affinity of KIV-lO/W72R compared

to that of the Thr66 and Met66 variants (Klezovitch & Scanu, 1996), its LigBS is of

127

Table 6-5: Probable conserved hydrogen bonds (A) in LigBS of various KIV-10.

 

 

KIV- 1 0/ KIV-1 0/ KIV-IO/ KIV-IO/
Met66 Met66/ Thr66/ W72R
EACA EACA
Arg36-NH2 ProS4-O 3.00 a a 3.20
Asn53-OD1 Asp55-OD] 2.59 2.85 3.02 3.76
-001 -N 2.75 2.82 2.44 2.51
-ND2 Asp57-O 2.77 2.90 3.02 3.17
-ND2 Asn5-O 2.50 2.75 2.63 2.54
-0191 Trp62-NE] 2.51 2.74 2.70 3.01
Asp57-OD1 Tyr74-OH 2.88 2.41 2.87 2.44
-OD1 Thr58-N 2.64 2.68 2.59 2.46
-OD2 0.242/212/255b 3.60 3.06 2.96 c
Pro61-O Cys75-N 3.00 2.85 2.94 3.43c
-o Ow228/264/258b 2.72 2.74 3.23 c
Trp62-N Arg52-O 2.77 2.81 2.95 2.66
-o -N 3.50 3.11 3.06 3.45
Cys63-N Glu73-O 3.04 2.98 3.01 349°
-0 -N 3.03 2.99 3.15 337°
Phe64-N o..201/202/208/219b 2.73 2.88 2.74 2.58
-o Gln23-N 2.61 2.64 2.69 3.38c
Arg71-O Thr65-N 3.10 3.10 2.96 2.58
-NH1 Arg32-O 2.65 2.75 2.86 337°
Trp72-O o..222/249/239b 3.48 3.05 2.96 c
Tyr74-N o..219/237/306b 2.72 2.96 2.88 c
-o Vall7-N 2.88 3.00 3.06 3.68c

 

a - Arg36 undergoes a conformational change.

b - Numbering of water molecules in respective structures.

c - Due to the shrinkage of the c-axis of the mutant, the solvent content is reduced to
22% (total 41 water molecules, Table 6—3) resulting in the loss of hydration interactions

and possibly some hydrogen bonds.

128

Table 6-6: Probable hydrogen bonds (A) in LigBS of KIV dependent on ligand binding
and the Trp72Arg mutation.

 

 

Kringle Ligand KIV-1 0/ KIV-10/ KIV-10/
Met66/EAC A Thr66/EACA W72R
Arg36-NE EACA-OZ 2.98 2.72 -
-NH1 Ow242/291 3.01 3.12 -
-NH2 EACA-OZ 3.05 2.87 Pr054-O (3.20)
-NH2 Arg32-N111a - 2.67 3 .22
-NH2 Arg32-NH? - - 3.25
Pr054-O Ow261/244 2.97 2.98 Arg36-NH2 (3.20)
Asp55-OD2 EACA-NZ 2.62 2.75 Arg32-NE (3.16)ll
Asp57-OD2 EACA-NZ 2.53 2.87 Arg72-NH2 (3.01)
-NE EACA-O 2.65 3.13 -
Arg7l-NH1 EACA-O 3.00 2.75 -
-NH2 A - - Thr29-OG1 (3.20)a

 

a - Symmetry related molecule.

129

 

 

/ I
Asp57
Trp62
. Q
c.39 . Asp55 11:0\\ e.
‘\ ’5‘“ \
" .‘ 9 if}? 0‘
351:. \ Phe64
{- '19, i 3" '5
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Arg32

Figure 6-2: Stereoview of final (2Fo-Fc) electron density of EACA of KIV-
10/I'hr66/EACA displaying the embedded binding mode. Contoured at the 10 level.

Two conformations of Arg32 are shown. Arg32 of symmetry related molecule (Arg132)

is in red.

130

special interest. Substituting a non-aromatic amino acid for Trp72, an obviously
important residue of the hydrophobic pocket along with Trp62 (Figure 6-3), would in
itself be expected to lead to some impairment of binding affinity. The structure of KIV-
lO/W 72R additionally shows that Arg72 extends across the LigBS toward the negatively
charged Asp55/Asp57 pair making a hydrogen bonded salt bridge with Asp57 (Table 6-6)
and thusly obstructs access to the LigBS. Since the cationic center consisting of Arg36
and Arg71 does not undergo any structural changes and is positionally similar to that of
the unliganded KIV-10 molecule (Figure 6-1 & 6-3), the arginyl of Arg72 occupying the
binding groove of the LigBS appears to be the structural deterrent responsible for the lack
of binding affinity of the KIV-10 mutant.
All four KIV-10 variants crystallized in the orthorhombic space group P212121.
Three of the crystals are isomorphous. while the c-axis of the fourth, KIV-IO/W72R, is
about 7.0 A shorter (Table 6-3). This 10% shrinkage is the result of different molecular
packing of the KIV-IO/W 72R molecules along the c-axis of the crystal. Superposition of
the mutant with any of the three isomorphous variants showed very little rotation of the
molecules with respect of each other. with only a translational difference along the z-
direction. In all four crystal arrangements, kringle molecules form infinite chain columns
parallel to the y-direction with a 15 A wide channel between chains. In KIV-10/W72R,
two neighboring symmetry related molecules are aligned approximately 3.5 A closer
along the crystallographic c-axis. while the packing along the two other axes remains
about the same. The net result is to shrink the channel by about 7 A at the expense of
solvent water molecules. The solvent fraction of the three isomorphous KIV-10

structures is approximately 30.6 "/0 (VM =1.76 A3/Da). In the case of the W72R mutant,

131

 

Figure 6-3: Superposition of KIV-IO/W 72R (dark gray) and KIV-IO/Met66/EACA (light

gray) showing Arg72 approximating EACA binding. Note different conformation of

Arg36 in the two structures.

132

the shrinkage along the c-axis decreases the solvent content to 22.0 % (VM =l.56
A3/Da). The number of water molecules found in the four different kringle structures
varies from 41 to 109 and is generally related to the resolution of the diffraction data but

in the case of KIV-10/W72R, it depends primarily on the solvent fraction of the crystals.

IV. DISCUSSION

The overall topology of human KIV-10 is an oblate ellipsoid of approximately 28 A
in diameter and 18 A thick (Figure 6-1). The LigBS of KIV-10 is similar to those of
plasminogen K1 and K4 (Mulichak et al., 1991; Wu et al., 1991; Mathews et al., 1996).
All three key segments, the anionic, cationic and hydrophobic centers, are essentially
positioned optimally for ligand binding in KIV-10/Met66 and except for Arg36 (Figure
6-1), remain so on ligand binding in KIV-lO/Met66/EACA and KIV-lO/Thr66/EACA.
In KIV-IO/Met66, residue Asp55 is in the same orientation (X1 z 60°) as in plasminogen
K1 and K4.

All three binding centers are operative in KIV-IO/Met66/EACA and KIV-
lO/Thr66/EACA examined in the present work and EACA binds to the LigBS in the
embedded way (Figures 6-1 & 6-2). This binding mode, also observed in plasminogen
K1 and K4 (Figure 6-1), is consistent with binding constants of different ligands
discussed earlier. Thus, the binding of embedded KIV-IO/I‘hr66/EACA is very different
from the unbounded mode previously reported for the KIV-10/Thr66/EACA structure
(Mikol et al., 1996). It is noteworthy, therefore, that lysine binding kringles can bind

ligand in two different ways.

The source of the binding mode variance between our work and that of Mikol et al.,
(Mikol et al., 1996) most likely resides in the different conditions used for crystallization.
The major differences are: unbounded KIV-10/Thr66/EACA, protein concentration 5
mg/mL, pH 7.5, in the presence of 40 mM NaCl; present work KIV-10H" hr66/EACA,
protein concentration 12 mg/mL. pH 5.6, no NaCl but with 10% 2-propanol and a lO-fold
molar excess of EACA (latter unreported in the Sandoz crystallization). Although the
different pH conditions might seem important. liganded K1 and K4 crystals in the
embedded mode were grown at higher pH of 6.5 and 6.0, respectively. It is of some note
that in the LigBS of unliganded plasminogen K1 crystals, there are two chloride ions in
the vicinity of the cationic center, which appear to interact with and neutralize Arg35 and
Arg71 electrostatically (Wu et al., 1994). In the case of unliganded plasminogen K4, the
anion is a sulfate and the interaction is more complicated by the additional presence of a
symmetry related molecule (Mulichak et al., 1991). Thus, it is conceivable that the
unbound ligand binding mode of KIV-10/Thr66/EACA (Mikol et al., 1996) could result
because of an anion compensated cationic center. However, examination of the B-values
of the water molecules in the LigBS of the latter reveals only evidence for water
molecules of solvation (no inordinately small B-values suggesting anions).

The unique features of the LigBS of the apo(a) KIV-10 of rhesus and chimpanzee are
the point mutations that lead to an impairment EACA binding and binding to lysine-
sepharose (Scanu et al., 1993; Klezovitch & Scanu, 1996; Chenivasse et al., 1998). The
structure of the recombinant mutant KIV-10/W72R shows that Arg72 extends along the
ligand binding grove parallel to the expected position of EACA, toward the negatively

charged Asp55/Asp57 anionic center and makes a hydrogen bonded salt bridge with

134

 

Asp57 (Figure 6-3, Table 6-6). Thus, the Arg72 side chain mimics ligand binding and
loss of binding ability is the result of a steric blockage of the LigBS by Arg72 physically
occupying part of the site. The Li gBS structure of KIV-10/W72R is similar to the LigBS
region of PT-Kl, which has an Arg72 equivalent but lacks the Asp57 to make a salt
bridge. In this case, Arg72 appears to form a salt bridge with Asp55. This structure was
used previously to closely model the LigBS of rhesus KIV-IO/W72R (Scanu et al., 1993).
In turn, the Asp57Asn point mutation in the LigBS of KIV-10/Asn57 of chimpanzee
simply lowers the negative electrostatic charge of the anionic center that appears to be so
critical for the binding of the quaternary, positively charged s-amino group of lysine and
other zwitterionic ligands. This only leads to an electrostatic impairment of an otherwise
capable LigBS. Since the Asn57 side chain is geometrically nearly isostructural with the
Asp57, which it replaces and can be a hydrogen bond donor or acceptor, it would seem
that the LigBS function of KIV-10 of chimpanzee should be less impaired compared to
that of the rhesus monkey, primarily because the steric impairment of the latter excluded
ligand physically. This suggested difference in LigBS function between the KIV-10 of
rhesus and chimpanzee could not be assessed further because the lysine binding
conditions were not provided for the chimpanzee product.

Superposition of the KIV-10/Met66 and the ligand bound KIV-IO/Met66/EACA and
KIV-lO/Thr66/EACA structures shows that the binding of EACA in KIV-10 is
accompanied by: (1) a displacement of water molecules from the EACA binding groove
and (2) a movement of the arginyl residue of Arg36 (Figure 6-1). Three water molecules
(0,258, 09,259, 0,9260) extending from the anionic to the cationic center in KIV-

10/Met66 are displaced by EACA with ligand binding. Two other hydrogen bonding

135

water sites, located in the LigBS region but not in the EACA binding groove, are
conserved in both structural types (Table 6-6). Similar to binding of EACA, the
Trp72Arg mutation causes the displacement of water molecules from the binding groove.
Additional examination of the residues within 5.0 A of the LigBS indicates only a
conformational change in residue Arg36 on binding EACA. In the ligand-free structures
of KIV-10/Met66 (Figure 6-1) and KIV-IO/W72R (Figure 6-3), residue Arg36 adopts an
extended conformation, almost parallel to the LigBS groove, and utilizes a hydrogen
bond with Pr054-O (Table 6-5). Upon ligand binding, Arg36 of the Met66 and Thr66
KIV-10 variants swings around the CA-CB bond by about -120° toward the cationic
center, but remains extended, bringing the guanidinium group close to the carboxylate of
EACA to assist Arg71 in stabilizing the anionic end of the ligand as in plasminogen K4
(Figures 6-1, 6-2 & 6-3, Table 6-6). The movement is accompanied by an approximately
1.3 A shift of the tyrosyl ring of the sidechain of Tyr4l into the vacancy created by the
Arg36 conformational change. When Arg36 is in the ligand binding posture, two water
molecules (Ow26l, Ow244, Table 6-6) also occupy the vacancy created by the shift. The
KIV-10/Thr66/EACA also shows the arginyl reorientation but the guanidinium amino
groups correspond to weaker electron density so their orientation is not as certain as that
of the Met66 variant.

Superposition of the KIV-10 variants also revealed different conformations for
Arg32. This residue is located on the surface of the kringle and is adjacent to the LigBS
of a symmetry—related molecule and appears to play a role in molecular packing. The
conformation of Arg32 is similar in the KIV-IO/Met66 and KIV-IO/Met66/EACA

structures. In addition to a favorable hydrogen bonding interaction between its carbonyl

 

oxygen atom and Arg71-NH1 in the free and ligand bound structures (Table 6-5), the

terminal guanidino nitrogen atoms of Arg32 in KIV-10/Met66/EACA participate in a
complicated electrostatic cluster comprised of the carboxyl group of EACA of the
symmetry related molecule and Arg36/Asp55 of the corresponding cationic and anionic
binding centers. Apparent charge neutralization is achieved by the cluster through an
pseudo cyclic arrangement of positive and negative centers (Arg32 of a neighboring
kringle, Asp55, quatemary amine of EACA. the carboxylate of EACA and Arg36 (Figure
6-2). In the KlV-lO/Thr66/EACA structure Arg32 has two alternate conformations,
which have been refined to an occupancy of 0.6 (extended) and 0.4 (bent). The Arg32 in
the extended conformation interacts with a symmetry related kringle unit (Figure 6-2),
whereas in the bent conformation it forms a hydrogen bond with cis Pr030-O (3.00 A) of
the same molecule. An additional water molecule participates in the stabilization of both
the extended and the bent conformation. In the KlV-lO/W72R mutant, Arg32 is in an
intermediate bent conformation displaying both intermolecular and intramolecular
interactions while bridging two kringle molecules. The NH] and NH2 atoms interact
with cis Pro30-O as in KIV-IO/Met66 and KIV-10/Met66/EACA but in addition, there
are intermolecular contacts between Arg32-NE and Asp55-OD2 (3.16 A) as well as
Arg32-NH2 and Arg36-NH2 (3.25 A) of the LigBS of a symmetry related molecule.

The current crystallographic studies of a wild type (Trp72) and mutant (Arg72) of
human apo(a) KIV-10 provide a detailed account of the way apo(a) KIV-10 binds to
lysine and its analogs. This information is of biological relevance in that lysine binding
and related activities of Lp(a) depend on the function of LigBS of KIV-10 (Scanu et al.,

1993; Scanu et al., 1994). The kringle. according to current dogma, is considered to be

137

 

exposed because it has no apparent formal linkages with the lipoprotein component of
Lp(a). In keeping with this notion is the lysine binding deficiency of rhesus monkey and
the human mutant Lp(a) having the critical Trp72 replaced by Arg in the LigBS of KIV-
10. From the standpoint of the athero-thrombogenic potential of Lp(a), this type of
mutation has been suggested to be benign on the premise that it would be unable to
interfere with the plasminogen to plasmin conversion (Scanu et al., 1993; Scanu et al.,
1994). This view has recently received support from studies in transgenic mice (Hughes
et al., 1997). Because of the pathobiological significance of the LigBS and its potential
for mutability, it would be of further interest to study additional natural and/or artificial
mutations in order to define their impact on the lysine binding activity of apo(a) KIV-10.
In the current study we have shown that the replacement of Met by Thr in position 66 had
no significant structural consequences in agreement with the finding that this common
human mutation is functionally silent. The example provided by chimpanzee Lp(a) of a
substitution at Asp55 by Asn is of interest in that we predict that such a substitution
would lead to only a relatively modest degree of a functional impairment of lysine
binding function compared to that of the rhesus. A functional hierarchy in the LigBS
might account for the variability in lysine binding activity among human subjects based
on measurements in the whole plasma (Scanu et al., 1994; Hoover-Plow et al., 1996). In
this context, however, we cannot rule out the contribution to the binding by a second
LigBS contained in the region between apo(a) KIV-5 and KIV-8 (Edelstein et al., 1995).
This region is usually open in free apo(a) whereas it is essentially masked when apo(a) is
a constituent of the Lp(a) particle (Edelstein et al.. 1995; Ernst et al., 1995). A structural

understanding of the region may be of some consequence in view of its suggested

138

 

 

 

inv

1994

involvement in the lysine-mediated binding of apo(a) to fibrinogen (Klezovitch et al.,

1996).

139

Angiogenesis

Arteries

Atherosclerosis

Blood Coagulation

Cascade

Blood Clotting
Cerebrovascular Disease
Coagulation Cascade

Denaturation

Differentiation

Dyne

 

GLOSSARY

formation of new blood vessels that occurs as a result of
the growth of capillaries by vascular sprouting from pre-
existing vessels

large blood vessels that lead blood away from the

heart.

deposition of fatty compounds on the inner lining of the
coronary arteries or any other arteries. The originally
smooth lining of the artery becomes roughened as the
atherosclerotic plaque collects in the artery.

series of stepwise reactions that utilize the protein
molecules circulating in the blood in order to seal an area
of endothelial damage and to stop blood loss.

see Blood Coagulation Cascade.

disruption of the normal blood supply to the brain

see Blood Coagulation Cascade

Structure alternations in proteins caused by heat, extreme
pH or by exposure to organic solvents, detergents or
certain solutes.

change in structure and function of a cell as it matures
force necessary to give acceleration of one centimeter per

second per second to one gram of mass.

140

 

 

En

Er

Fil

Ht

In

Endothelium

Epithelium

Fibrinolysis

Hemorrhage

Hemostasis

Hypertension

Infarction

Kringle domain

Megakaryocyte

Metastasis

Occlusion

 

type of epithelium composed of a single layer of smooth
thin cells that lines the heart, blood vessels, lymphatics
and serous cavities.

any tissue which covers a surface or lines a cavity and
which performs protective, secreting or other functions.
phenomenon of clot dissolution.

discharge of blood from a ruptured blood vessel.
stoppage of bleeding or the stoppage of the circulation of
blood in a part of the body.

high blood pressure.

area of dead myocardial tissue. The severity of a
myocardial infarction (also known as a heart attack)
depends on the size of the artery that is blocked and the
extend of the blockage.

structural and functional modular unit of approximately
80 amino acids cross-linked by three disulfide bridges in
the characteristic pattern. Kringle motives are found in
various blood proteins.

platelet precursor formed in the bone marrow.

ability of tumor cells to leave their site of origin and
migrate to other locations in the body, where a new
colony is established.

closure of a blood vessel.

141

Platelet count

Primary structure

Quaternary structure

Secondary structure

Stem cell

Tertiary structure

Thrombocythemia

Thrombocytopathy

Thrombocytopenia

Thromboplastin

Thrombopoiesis

Thrombosis

 

number of platelets per cubic millimeter. Platelets
normally average between 140,000 and 400,000/mm3.
the order of amino acid residues in polypeptide chain.
the arrangement and nature of the binding of the subunits
and domains together.

the conformation of a polypeptide chain and the intra-
chain hydrogen binding scheme.

cell in bone marrow that gives rise to different types of
blood cells.

the folding of the polypeptide which results in a three-
dimensional structure.

increased platelet counts.

abnormal platelet functions in adhesion and aggregation.
decreased platelet counts is the most common platelet
disorder of insufficient platelet production and/or
excessive platelet removal from circulation.

membrane glycoprotein and a clotting factor that is
located in the tissue adventita and comes into contact
with blood after a vascular injury. In combination with
FVII/FVIIa and calcium, it initiates the blood
coagulation cascade.

production of platelets

condition of clot formation

142

 

Names, three- and one-letter codes and molecular weights of the common amino acids

 

 

 

 

 

 

 

 

 

 

 

 

Amino Acid Three- and one- Mr. Amino Acid Three- and one- Mr.
letter code letter code

Glycine Gly G 75 Alanine Ala A 89
Valine Val V 1 l7 Leucine Leu L 13 1
Isoleucine Ile I l 3 l Phenylalanine Phe F 165
Tyrosine Tyr Y 1 8 1 Tryptophan Trp W 204
Serine Ser S 1 05 Threonine Thr T 1 l9
Cystein Cys C 121 Methionine Met M 149
Asparagine Asn N l 3 2 Glutamine Gln Q 146
Aspartic acid Asp D 133 Glutarnic acid Glu E 147
Lysine Lys K 146 Arginine Arg R l 74
Histodine His H 1 5 5 Proline Pro P 1 15

 

 

 

 

 

 

 

 

143

 

 

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