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.Cell wall-anchored proteins play critical roles in the virulence of most Gram-positive bacterial pathogens by acting as adhesins or invasins and/or interfering with various arms of the host innate or specific immune defenses. The vast majority of these virulence proteins are retained at the bacterial surface after secretion by a mechanism that involves the covalent linkage of target proteins to the peptidoglycan layer of the cell wall. This linkage is catalyzed by membrane-associated transpeptidases called sortases (1, 2). 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 sortase-mediated transpeptidation reaction is also being increasingly used in a variety of biotechnology applications (5-8).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 (where X is any amino acid), followed by a hydrophobic region and a tail of positively charged residues that locates the substrate to the cell surfacefollowingsecretion(2,9).Inonecurrentmodelofsortasedependent transpeptidation, the LPXTG motif is specifically recognized by the enzyme (10), and the thiolate group of an essential active sit...

The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modelling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.

The Staphylococcus aureus sortase transpeptidase SrtA isoform is responsible for the covalent attachment of virulence and colonization-associated proteins to the bacterial peptidoglycan. SrtA utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(epsilon-Gly(5))-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved C-terminal LPXTG sequence. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly(5) portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we describe the development of a general in vitro method for elucidating the substrate specificity of sortase enzymes. In addition, using immunofluorescence, cell adhesion assays, and transmission electron microscopy, we establish links between in vitro substrate specificity and in vivo function of the S. aureus sortase isoforms. Results from these studies provide strong supporting evidence of a primary role of the SrtA isoform in S. aureus adhesion and host colonization, illustrate a lack of specificity cross talk between SrtA and SrtB isoforms, and highlight the potential of SrtA as a target for the development of antivirulence chemotherapeutics against Gram-positive bacterial pathogens.

Several small molecule vinyl sulfones were found to exhibit irreversible time-dependent inhibition of the Staphylococcus aureus sortase SrtA in vitro. A representative of these compounds was shown to impair the ability of S. aureus bacteria to bind fibronectin-coated surfaces through in vivo inhibition of SrtA-mediated linkage of fibronectin to the cell surface. These data highlight the potential use of small molecule vinyl sulfones as chemotherapeutics to prevent adhesion to and colonization of host tissues during S. aureus infection.