Anchor Structure of Staphylococcal Surface Proteins

Abstract

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the amino of the pentaglycine cross-bridge in the staphylococcal peptidoglycan. We asked whether antibiotic cell wall synthesis inhibitors interfere with the anchoring of surface proteins. Penicillin G, a transpeptidation inhibitor, had no effect on surface protein anchoring, whereas vancomycin and moenomycin, inhibitors of cell wall polymerization into peptidoglycan strands, slowed the sorting reaction. Cleavage of surface protein precursors did not require a mature assembled cell wall and was observed in staphylococcal protoplasts. A search for chemical inhibitors of the sorting reaction identified methanethiosulfonates and p-hydroxymercuribenzoic acid. Thus, sortase, the enzyme proposed to cleave surface proteins at the LPXTG motif, appears to be a sulfhydryl-containing enzyme that utilizes peptidoglycan precursors but not an assembled cell wall as a substrate for the anchoring of surface protein.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

“…We propose that the LPXTG motif within sorting signals of secreted proteins is cleaved by the sulfhydryl group of C184 located within this pocket. Consistent with this hypothesis is the strict conservation of H120 and C184 within the putative active site, the detrimental effects of a C184A mutation (14), and the observation that methyl methanethiosulfonate (43), a compound preferentially reactive with thiolate ions (44), abolishes sortase activity (9).…”

“…The carboxyl group of threonine is amide-linked to the amino group of the pentaglycine crossbridges, thereby anchoring the C-terminal end of surface proteins to the staphylococcal cell-wall peptidoglycan (5,6). It seems that lipid II [undecaprenyl pyrophosphate-MurNAc(-L-Ala-D-iGln-L-Lys(NH 2 -Gly 5 )-D-Ala-D-Ala)-␤1-4-GlcNAc)], a membrane-anchored precursor of cell-wall synthesis (7,8), serves as a substrate for surface protein anchoring (9). Surface proteins tethered to lipid II may subsequently be incorporated into the peptidoglycan by means of the transglycosylation and transpeptidation reactions of bacterial cell-wall synthesis (10).…”

Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus

“…Purified recombinant sortase lacking its N-terminal signal peptide/membrane anchor catalyzes the transpeptidation reaction of surface protein anchoring in vitro, using LPXTG peptides and NH 2 -Gly 3 as a peptidoglycan substrate (9). Both the in vivo and in vitro reactions of sortase can be inhibited with the thiolate reagent methylmethane thiosulfonate (for example MTSET) 2 or parahydroxy-mercurybenzoic acid (12), but not with sulfhydryl reagents such as iodoacetate or iodoacetamide (7,9). Surface proteins bearing LPXTG motifs as well as sortase genes have been found in many Gram-positive bacteria, and the presence of a single conserved cysteine within a LXTC signature sequence is a distinguishing feature of these enzymes (13,14).…”

“…During cell wall anchoring, surface proteins are cleaved between the threonine and the glycine of the LPXTG motif (3) and subsequently amide-linked to the pentaglycine cross-bridges of the cell wall peptidoglycan (4 -7). Lipid II, the biosynthetic precursor of cell wall synthesis, is presumed to act as the peptidoglycan substrate of sortase (7). 1 The lipid-linked surface protein intermediate is then incorporated into the peptidoglycan via the transpeptidation and transglycosylation reactions of bacterial cell wall synthesis (7).…”

“…Lipid II, the biosynthetic precursor of cell wall synthesis, is presumed to act as the peptidoglycan substrate of sortase (7). 1 The lipid-linked surface protein intermediate is then incorporated into the peptidoglycan via the transpeptidation and transglycosylation reactions of bacterial cell wall synthesis (7). Staphylococcus aureus sortase (SrtA), a 206-amino acid transpeptidase with an N-terminal transmembrane anchor/signal peptide (8), catalyzes the anchoring of surface proteins to peptidoglycan (9,10).…”