title: Clostridium difficile cell wall protein CwpV undergoes enzyme-independent intramolecular autoproteolysis.
abstract: Clostridium difficile infection is a leading cause of antibiotic-associated diarrhea, placing considerable economic pressure on healthcare systems and resulting in significant morbidity and mortality. The pathogen produces a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologs. CwpV is the largest member of this family and is expressed in a phase-variable manner. The protein is post-translationally processed into two fragments that form a noncovalent, heterodimeric complex. To date, no specific proteases capable of cleaving CwpV have been identified. Using site-directed mutagenesis we show that CwpV undergoes intramolecular autoproteolysis, most likely facilitated by a N-O acyl shift, with Thr-413 acting as the source of a nucleophile driving this rearrangement. We demonstrate that neighboring residues are also important for correct processing of CwpV. Based on protein structural predictions and analogy to the glycosylasparaginase family of proteins, it appears likely that these residues play key roles in determining the correct protein fold and interact directly with Thr-413 to promote nucleophilic attack. Furthermore, using a cell-free protein synthesis assay we show that CwpV maturation requires neither cofactors nor auxiliary enzymes.
authors: Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.
authors: Marcin
authors: M
authors: Dembek
authors: Catherine B
authors: CB
authors: Reynolds
authors: Neil F
authors: NF
authors: Fairweather
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins; C070683:surface layer protein A, Bacteria