Intramembrane proteases (IPs) regulate diverse signaling pathways in all three domains of life by cleaving membrane-associated substrates. Currently, there are four known IP families, including intramembrane metalloproteases (IMMPs), which contain characteristic HEXXH and NPDG motifs that coordinate a zinc ion at the active site. The study of IMMPs, including RseP (Escherichia coli) and SpoIVFB (Bacillus subtilis) have provided insights into potential mechanisms for substrate interaction and cleavage, which could guide efforts to develop inhibitors of IMMPs that regulate virulence in bacterial pathogens. Work described in this dissertation has advanced the knowledge of inhibition and substrate interaction of SpoIVFB, which cleaves Pro-σK during endosporulation. Improved methods are presented for heterologous production in E. coli of SpoIVFB, Pro-σK, and the inhibitory proteins BofA and SpoIVFA. Three conserved BofA residues (N48, N61, T64) in or near transmembrane segment (TMS) 2 are required for SpoIVFB inhibition. Cross-linking indicates that BofA TMS2 occupies the SpoIVFB active site region. Interestingly, BofA and SpoIVFA do not prevent interactions between portions of Pro-σK and SpoIVFB, so all four proteins can exist in a complex. A structural model of SpoIVFB with BofA and parts of SpoIVFA and Pro-σK was built using partial homology and constraints from cross-linking and co-evolutionary analyses. The model suggests that SpoIVFA stabilizes BofA in the SpoIVFB active site region and leads us to propose that BofA TMS2 sterically hinders access of the substrate. Our work has also advanced knowledge of interactions between Pro-σK and the broadly conserved SpoIVFB NPDG motif, which is located in a predicted short loop that interrupts TMS4 and faces the active site. Three highly conserved residues (N129, P132, P135) of SpoIVFB were found to be important for substrate interactions and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. More work is needed to fully understand how IMMPs interact with their substrates and whether the insights from BofA inhibition of SpoIVFB can be applied to other IMMPs. Outstanding questions and future directions related to these two projects are described.
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