Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition

3D Structure of the Transient Intermediate of the Enzyme–Substrate Complex of Sortase A Reveals How Calcium Binding and Substrate Recognition Cooperate in Substrate Activation

“…Recent crystal structures of Streptococcus pyogenes sortase A (SpySrtA) complexed with LPATA or LPATS show good agreement with the present NMR structure of the thioester intermediate, although the SpySrtA complex was devoid of covalent bonds (the active-site residue C208 being mutated to Ala). 37 Similarities include the side chain of the Thr residue of the substrate deep inside the recognition pocket A and the absence of a direct contact with the arginine residue near the active site (R216 in SpySrtA). Different from S. aureus SrtA, a greater intrinsic binding affinity for substrate peptide was reported for SpySrtA, which has no calcium binding motif.…”

“…The structural details of the thioester intermediate have been unknown, leaving the mechanisms of substrate recognition and the subsequent reaction that generates a stable amide bond subject to speculation. In addition, structure-based drug discovery of antibiotics targeting SrtA is difficult without detailed knowledge of the 3D structures involved in the enzyme mechanism. , While several structures of SrtA and its complexes with substrate analogues and non-covalently bound peptides have been determined, − it is not clear how well these represent the structure of the actual intermediate complex with the substrate (Figure ). In previous work, we showed that the authentic thioester intermediate can be produced at low concentrations and, using pseudocontact shifts (PCS) generated by two site-specifically attached lanthanide tags, succeeded in determining the 3D structure of SrtA in the intermediate despite its instability and the presence of a large excess of free SrtA .…”

A unique binding mode of P1′ Leu-containing target sequences for Streptococcus pyogenes sortase A results in alternative cleavage

“…Wild-type (WT) spySrtA (residues 81–249, corresponding to PDB ), WT lmSrtA (corresponding to residues 71–222, UniProt ID SRTA_LISMO, PDB ), H143A spySrtA, I211P spySrtA, C208A spySrtA, and the β7–β8 loop chimeric proteins, spySrtA monocytogenes and lmSrtA pyogenes sequences were expressed using the pET28a(+) plasmid (Genscript) in Escherichia coli BL21 (DE3) cells, as described previously. 5,22,23,25,27 All plasmids contained a 6xHis tag followed by TEV cleavage site (sequence: ENLYFQS). The purification protocols were also those used previously.…”

“…The purification protocols were also those used previously. 5,23,25,28 Briefly, following induction by IPTG and overexpression, the cells were harvested in lysis buffer [0.05 M Tris pH 7.5, 0.15 M NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA)], lysed via sonication, and clarified using centrifugation, followed by filtration of the supernatant. Initial purification was conducted via immobilized metal affinity chromatography (IMAC) using a 5 mL HisTrap HP column (Cytiva), with wash [0.05 M tris pH 7.5, 0.15 M NaCl, 0.02 M imidazole, 0.001 M TCEP] and elution [wash buffer with 0.3 M imidazole] buffers.…”

“…These enzymes catalyze a transacylation reaction that attaches proteins, including toxins and environmental sensors, to the peptidoglycan layer at the cell surface. 1–5 The first sortase enzyme, Sortase A (SrtA) from S. aureus , was discovered in 1999. 4,6 This enzyme is selective for the cell wall sorting signal (CWSS) sequence LPXTG, where X = any amino acid.…”