Staphylococcus aureus Sortase A Transpeptidase

Abstract

Many virulence factors in GramSurface proteins on bacteria are frequently virulence factors, promoting bacterial adhesion, resistance to phagocytic killing, and host cell invasion during infection. In Gram-positive bacteria these proteins are often covalently anchored to the cell wall by sortase enzymes, a family of novel cysteine transpeptidases (1-3). The sortase A protein (SrtA) 2 from Staphylococcus aureus has been characterized extensively (4) and anchors proteins bearing a cell wall sorting signal that consists of a conserved LPXTG motif (where X is any amino acid), a hydrophobic domain, and a tail of mostly positively charged residues (4 -6). SrtA cleaves in between the threonine and glycine of the LPXTG motif (7) and catalyzes the formation of a peptide bond between the carboxylgroup of the threonine and the amine-group of the cell-wall precursor lipid II (7-9). The lipid II-linked protein is then incorporated into the peptidoglycan of the cell wall via the transglycosylation and transpeptidation reactions of bacterial cell-wall synthesis. Sortases represent an attractive target for new anti-infective agents, because they are widely distributed among a variety of bacterial pathogens (10, 11) (e.g. Bacillus anthracis, Listeria monocytogenes, Streptococcus pneumoniae, and Streptococcus pyogenes), and have been shown to be required for virulence (12-16).The catalytic domain of SrtA (SrtA ⌬N59 , residues 60 -206) adopts a conserved eight-stranded ␤-barrel fold (17, 18). The active site is organized around the catalytically essential side chain of Cys-184, whose thiolate nucleophilically attacks the threonine carbonyl carbon within the LPXTG sorting signal, forming a thioester linkage between the enzyme and substrate (19). In addition to Cys-184, the hydrophilic side chains of His-120 and Arg-197 are absolutely required for catalysis (20 -22). These residues likely participate in general acid/base catalysis, and one of them must activate the thiol for nucleophilic attack, because it is protonated at neutral pH (23). The indole ring of Trp-194 partially shields the cysteine thiol from the solvent, and its mutation to alanine reduces enzyme activity 4-fold through an unknown mechanism (20). Using NMR and crystallography, the LPXTG sorting signal binding site has recently been localized to a surface formed by strands ␤4 and ␤7, and to a proximal loop that connects strands ␤6 to ␤7(the ␤6/␤7 loop) (18,22). Substrate binding may occur through an induced-fit mechanism involving conformational changes in the ␤6/␤7 loop, because it is disordered in the absence of the sorting signal substrate (17,18). Ca 2ϩ stimulates the activity of SrtA ⌬N59 in vitro (17) and may enable S. aureus to increase the rate of surface protein anchoring as it encounters elevated concentrations of this ion at sites of infection. Because many surface proteins function as virulence factors, the stimulatory effect of Ca 2ϩ likely plays an important role in the infection process. Previously we showed that Ca 2ϩ bound to an ordered pocket positio...

The binding mechanism, multiple binding modes, and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

“…This is consistent with an induced-fit mechanism. However, the observed fluctuations of the b6/ b7 loop between open and semi-closed states, and the previously reported NMR data on micro to millisecond timescale dynamics of these same residues, 30 point toward a conformational selection mechanism. Taken together, experiments and simulations therefore suggest a recognition model of conformational selection followed by induced fit, consistent with the emerging paradigm for a diverse range of recognition processes.…”

“…20 Because it has been shown to be important for the catalytic activity of the enzyme, a calcium ion was added to the structure, positioned in its known binding site. 20,30 Initial coordinates for the holo SrtA simulations were generated from the 2KID NMR structure in which an LPAT analog is covalently attached to the catalytic C184 residue in the enzyme's active site. 21 For the holo structure, an unmodified LPATG sequence was subsequently docked into this site using Glide with the XP scoring function, 42 and the structure in which the positions of the L, P, and A residues most closely matched those in the NMR structure was chosen for simulation.…”

“…However, interaction of the b6/b7 loop with the calcium ion was limited to a contact with residue E171 that was more stable than in the apo simulations and did not include interaction with residue D170, in agreement with the previous site-directed mutagenesis experiments. 30 …”

Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

“…Recent NMR analyses by Naik et al (38) revealed that the backbone dynamics of the ␤6/␤7 loop in SrtA change significantly upon calcium binding. 15 Engineering the Substrate Specificity of S. aureus SrtA used the NPQTN-containing peptide Abz-KVENPQTNAGTDap(DNP)-NH 2 .…”

Crystal Structure of Streptococcus pyogenes Sortase A

“…In addition to providing the first molecular structure of a sortase enzyme (18), NMR has also been used to probe the dynamics of Sa-SrtA and determine the overall binding orientation of the sorting signal peptide to the protein (24,25). These studies revealed that Ca 2ϩ acts as an allosteric activator by stabilizing the structure of the ␤ 6 /␤ 7 loop in Sa-SrtA in a closed, substrate binding state and that the binding site for the LPXTGsorting signal comprises residues from ␤ 4 and␤ 7 and the ␤ 3 /␤ 4 and ␤ 6 /␤ 7 loops.…”