Structural and functional studies of suppressor of copper sensitivity proteins from Proteus mirabilis

Abstract

The correct formation of disulfide bonds is a crucial step in the folding of many virulence factors in bacteria. In many Gram-negative bacteria, redox machinery in the periplasm ensures this occurs efficiently and correctly for secreted and membrane proteins through the processes of disulfide bond oxidation and isomerisation. This machinery has been well characterised in Escherichia coli (strain K-12) where it consists of four key disulfide bond forming (Dsb) proteins, DsbA, DsbB, DsbC and DsbD. E. coli DsbA is the prototypical bacterial oxidase, which catalyses the formation of disulfide bonds in its protein substrates. When the active site cysteines of DsbA become reduced, DsbA needs to be reoxidised by its redox partner, DsbB, to continue to function. DsbA often forms disulfide bonds incorrectly and DsbC corrects this by isomerising or shuffling the disulfide bonds in the substrate protein. As DsbC is only active when its catalytic cysteines are reduced, it also has a redox partner, DsbD. Over the past 27 years, Dsb-like proteins from various bacteria have been studied and it has become clear that there is great diversity in the number and type of proteins that contribute to the redox machinery in different bacteria. Some bacteria have multiple Dsb proteins and distinct Dsb-like systems that have specific roles. The suppressor of copper sensitivity (Scs) proteins form one such system. Originally identified in Salmonella enterica serovar Typhimurium, the Scs locus encodes four proteins (ScsA, ScsB, ScsC and ScsD). The S. Typhimurium locus was found to confer copper tolerance to a copper sensitive E. coli mutant, so it is thought that these proteins play a role in bacterial response to copper stress. Three Scs proteins (ScsB, ScsC and ScsD) have predicted thioredoxin folds and catalytic cysteines, similar to the Dsb proteins. This thesis focuses on the characterisation of the ScsC and ScsB proteins from the uropathogen Proteus mirabilis. Prior to this work, ScsC had only been studied in two organisms, S. Typhimurium and Caulobacter crescentus. Chapter 2 is a Nature Communications paper that describes the genetic, biochemical and structural characterisation of P. mirabilis ScsC (PmScsC). This work revealed that PmScsC is a highly dynamic, trimeric disulfide isomerase that increases the swarming motility of P. mirabilis under copper stress. An N-terminally truncated PmScsC variant, PmScsCΔN, is monomeric and has little in vitro disulfide isomerase activity but is highly active as a dithiol oxidase. This variant could not complement the loss of ScsC in P. mirabilis, suggesting that PmScsC functions as a trimeric disulfide isomerase in vivo. The three crystal structures of PmScsC revealed that the region responsible for the highly dynamic nature of the protein is an 11 amino acid flexible linker between the trimerisation domain and catalytic domain. Replacement of this flexible linker with a rigid linker revealed that it was necessary for the disulfide isomerase activity of PmScsC. Chapter 3 describes the structural characterisation of the PmScsC variant, PmScsCΔN and the biochemical and structural characterisation of another variant PmScsCΔLinker. The structure of PmScsCΔN is very similar to the catalytic domains of the native PmScsC structures, suggesting that it is the lack of the trimerisation stem and thus change in oligomerisation state that converts it from an isomerase to an oxidase. To assess the role of the flexible linker in PmScsC, the linker residues (39-49) were deleted. This deletion mutant (PmScsCΔLinker) crystallised as a trimer but exists in different oligomeric states in solution and does not function as an oxidase or an isomerase. The characterisation of these variants adds to our understanding of the key structural features of PmScsC and their influence on the function of this trimeric disulfide isomerase. This chapter has been submitted to Acta Crystallographica Section D for publication.Based on work performed in C. crescentus, we hypothesised that the N-terminal periplasmic domain of PmScsB (PmScsBα) is the redox partner of PmScsC. Chapter 4 is a Journal of Biological Chemistry publication that describes the experiments performed to confirm this hypothesis. This publication also reports the crystal structure of PmScsBα, (the first ScsBα structure to be solved) and a SANS derived model of the complex formed between PmScsC and PmScsBα. I found that PmScsBα consists of two immunoglobulin-like domains, both of which interact with one PmScsC protomer in the SANS model.To gain more information about PmScsB, I wanted to produce the full-length transmembrane protein for further characterisation. Chapter 5 describes the work performed towards the design of robust expression and purification protocols for the production of PmScsB. In summary, this thesis describes the characterisation of PmScsC, PmScsC mutants, PmScsBα and the interaction between PmScsC and PmScsBα. It also contains the steps taken toward the production of the full-length transmembrane protein PmScsB for future analysis. This work adds to our understanding about the Suppressor of copper sensitivity proteins and the diversity of redox machinery in bacteria.