Crystal Structure of Streptococcus pyogenes Sortase A

Dewey G Mccafferty,Matthew L Bentley,Richard B Sessions,Richard K Hughes,Michael A Kehoe,Allister Crow,Wendy D Smith,Mark J Banfield,Jeff A Melvin,Paul R Race

doi:10.1074/jbc.m805406200

Dewey G Mccafferty, Matthew L Bentley + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m805406200

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Abstract

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.

Highlights

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces
The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis
The sorting signal that targets proteins for cell surface attachment is located at the C terminus of substrates and comprises a pentapeptide motif, typically LPXTG, followed by a hydrophobic region and a tail of positively charged residues that locates the substrate to the cell surface following secretion [2, 9]

Summary

Introduction

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. All Gram-positive pathogens express a “housekeeping” sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Proteins destined for cell-surface attachment contain a sorting signal recognized by these enzymes As this mechanism is unique to Gram-positive pathogens, inhibiting the reaction is an attractive target for the development of novel antibacterials [3, 4]. The Arg is thought to either stabilize the short-lived oxyanion-transition state [14] or to be involved in substrate recognition via a hydrogen bond [15] This reverse-protonation mechanism of catalysis, involving a Cys thiolate nucleophile and a His imidazolium general acid (but not a pre-organized ion pair), remains somewhat controversial as the pKa values of the Cys and His in Staphylococcus aureus sortase A (Sa-SrtA, the most extensively studied sortase enzyme) have been measured as ϳ9.4 and 6.2–7.0 [16, 17], respectively. Recombinant Sa-SrtA catalyzes the in vitro transpeptidation of LPXTG-containing peptides to pentaglycine [17, 21], the branched side chain of lipid II, the cell wall precursor to which sortase A substrates are attached in this organism [22, 23]

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