Hydrogen/Deuterium Exchange Mass Spectrometry and Site-Directed Disulfide Cross-Linking Suggest an Important Dynamic Interface between the Two Lysostaphin Domains

Abstract

Lysostaphin is a peptidoglycan hydrolase secreted by Staphylococcus simulans. It can specifically lyse Staphylococcus aureus and is being tested as a novel antibacterial agent. The protein contains an N-terminal catalytic domain and a C-terminal cell wall targeting domain. Although the two domains from homologous enzymes were structurally determined, the structural organization of lysostaphin domains remains unknown. We used hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and sitedirected disulfide cross-linking to probe the interface between the lysostaphin catalytic and targeting domains. H/DX-MS-mediated comparison of peptides from full-length lysostaphin and the separated domains identified four peptides of lower solvent accessibility in the full-length protein. Cross-linking analysis using cysteine pair substitutions within those peptides showed that two pairs of cysteines can form disulfide bonds, supporting the domain association role of the targeted peptides. The crosslinked mutant exhibited a binding capacity to S. aureus that was similar to that of the wild-type protein but reduced bacteriolytic activity probably because of restraint in conformation. The diminished activity was further reduced with increasing NaCl concentrations that can cause contractions of bacterial peptidoglycan. The lytic activity, however, could be fully recovered by reducing the disulfide bonds. These results suggest that lysostaphin may require dynamic association of the two domains for coordinating substrate binding and target cleavage on the elastic peptidoglycan. Our study will help develop site-specific PEGylated lysostaphin to treat systemic S. aureus infections.

Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans

“…Peptides that show a hydrogen/deuterium effect because of domain interaction are highlighted in red. The Cα atoms of residues that were replaced with cysteines and were cross-linked are shown as red balls 12. The catalytic and CWT domains are shown in yellow and green, the flexible linker is in brown.…”

“…Experimentally introduced cysteines in positions 332 (K332C) and 464 (T464C) can be cross-linked under oxidizing conditions 12, but the Cα carbon atoms of the mutated residues are > 45 Å apart in all four molecules in the structure and cannot even be approximated by a simple closure or hinge between lysostaphin domains (Fig. 7A).…”

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

“…Site-directed disulfide cross-linking was recently combined with HDX to predict the structural arrangement of the two domains of the peptidoglycan hydrolase lysostaphin [131].…”

Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats

“…Similarly, the formation of Cys-guanine-N7 (protein-DNA) conjugates in response to exposure of 1,3-butadiene, a component of vehicle exhaust and cigarette smoke, was identified by MS in over 150 proteins in human fibrosarcoma cells [135]. Lastly, HDX and cross-linking were recently employed in tandem to investigate tertiary structure in the non-cysteine-containing lysostaphin from Staphylococcus simulans [136]. First, HDX-MS was used to identify putative domain interfaces established by non-covalent interactions, then cysteine residues were engineered into the structure at positions predicted to be sufficiently close to promote disulfide bond formation [136].…”

“…Lastly, HDX and cross-linking were recently employed in tandem to investigate tertiary structure in the non-cysteine-containing lysostaphin from Staphylococcus simulans [136]. First, HDX-MS was used to identify putative domain interfaces established by non-covalent interactions, then cysteine residues were engineered into the structure at positions predicted to be sufficiently close to promote disulfide bond formation [136]. The successful formation of disulfide bonds, as judged by MS, provided key evidence of the proximity of the two regions in 3-dimensional space, illustrating that studies of protein structure by MS are achievable without amino acid labeling using reactive compounds.…”