Structural elucidation of the Cys‐His‐Glu‐Asn proteolytic relay in the secreted CHAP domain enzyme from the human pathogen Staphylococcus saprophyticus

Abstract

Cysteine peptidases (CP) are ubiquitous enzymes that play fundamental roles in many cellular metabolic pathways.1 In mammalian cells they are highly regulated in apoptotic pathways related to cancer and other severe disorders.2,3 In bacterial cell division, cell-growth and lysis peptidases are employed in the cleavage of peptidoglycans (autolysin).4,5 As secreted antigens and toxins, CPs are key to virulence in Gram-positive pathogens,6 and used to attack competing species in bacterial warfare.7 Viral genomes encode forms of the enzyme for the purpose of capsid trimming during maturation.8 CPs have been classified in clans based on their evolutionary relationships,9 in particular, the CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain (pfam: PF05257, MEROPS ID: C51) is classified as an endopeptidase and part of the CA clan of peptidases (CL0125). This 27-member superfamily includes synthetase/amidases, peptidases, viral proteinases, the NPLC/P60 families and other papain-related families.10 Pfam 22.0 lists 602 members for the CHAP domain family, including 475 from the bacterial kingdom (396 in the firmicutes phylum without any known 3D structure), 39 from eukaryota, and 81 from viruses and phage (predominantly involving Gram-positive phage). CHAP domains are often associated with other peptidases, bifunctional glutathionyl spermidine (GSP) amidases (type 2 and 3), choline binding, and with SH3 and/or Von Willebrand (VWA) domains to form multidomain systems that act cooperatively as versatile machineries for murein septum processing while anchored to the cell surface. Deletion of the CHAP-containing cse gene in Streptococcus thermophilus results in impaired separation of cells during mitosis, demonstrating its involvement in cell division.11 The staphylococcal phage ϕ11 hydrolase, a CHAP domain-containing enzyme, exhibits D-alanyl-glycyl endopeptidase and N-acetylmuramyl-L-alanyl amidase activity.12 In this note we present the solution NMR structure of CHAP domain encoded by gene SSP0609 of Staphylococcus saprophyticus [SWISS-PROT ID: Q49ZM2_STAS1; NESG target ID: SyR11]. S. saprophyticus, one of the three major human Gram-positive pathogens, possesses anchoring fimbriae that help colonize the urinary tract resulting in infection.13 Bacterial antigens are known virulence agents7, and SSP0609 is a secreted antigen with potential roles in the onset of S. saprophyticus infection. The protein comprises a type-I signal peptide in the N-terminal region (res. 1 - 47) and a globular CHAP domain in the C-terminal region (res. 48 - 155). The SSP0609 CHAP domain was found to have a highly-conserved Cys – His – Glu – Asn proteolytic relay active site.