Lysostaphin and LytM185-316 bind peptidoglycan or cell walls differently The involvement of different regions of lysostaphin in peptidoglycan binding has been investigated earlier. The results show that lysostaphin has affinity for the pentaglycine crossbridges themselves [34], but also binds cell

walls via the cell wall targeting domain [35]. In contrast, almost nothing is known about the role of different LytM fragments in peptidoglycan binding. Therefore, we investigated this question by the Stattic clinical trial Pulldown assay (Figure 4A). Comparing the amounts of protein in the pulldown and supernatant fractions, we found that the full length protein (LytM26-316) did not efficiently bind to peptidoglycan. Mutation of the Zn2+ ligand Asn117 to alanine, which should weaken the binding of the occluding region

to the catalytic domain, did not significantly change the situation. The TPCA-1 price isolated N-terminal domain of the enzyme also failed to bind to peptidoglycan, whereas LytM185-316 bound efficiently. When the Small molecule library two Zn2+ ligands His210 and Asp214 were separately mutated to alanine, the binding was lost again. Changing the third Zn2+ ligand, His293 of the HxH motif to alanine, made the protein insoluble as reported earlier [12], so that peptidoglycan binding could not be tested. The first histidine of the HxH motif, His291, is likely to act as a general base in catalysis [11]. When this residue was mutated to alanine, peptidoglycan binding was reduced, but not fully abolished. Figure 4 Pulldown assay of various LytM fragments and inhibitors with purified peptidoglycans from S. aureus . (A) Full length LytM and various fragments were analyzed by denaturing gel electrophoresis and Casein kinase 1 Coomassie straining either directly (control, C) or after separation into peptidoglycan binding (PG) and supernatant (S) fractions. (B) LytM185-316 was incubated with peptidoglycan in the presence of various protease inhibitors and the pellet fraction after pulldown analyzed by denaturing gel electrophoresis and Western blotting. The requirement of an intact active site for peptidoglycan binding was

also supported by inhibitor studies. We had previously shown that EDTA and 1,10-phenanthroline blocked activity, presumably by chelating Zn2+ ions. We now observed that both metal chelators also abolished binding of LytM185-316 to peptidoglycan (Figure 4B, lanes 1–2). In contrast, the weak Zn2+ ion chelator glycine hydroxamate and other small molecules and protease inhibitors did not interfere with peptidoglycan binding (Figure 4B, lanes 3–6). We conclude from these experiments that the accessibility and integrity of the active site is essential for the binding of the protein to peptidoglycan (Figure 4). Lysostaphin and LytM185-316 activities depend differently on pH Peptidoglycan hydrolase activities were assayed in a turbidity clearance assay, using S. aureus cells.