title: 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 site-directed 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 cross-linked 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.
authors: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
authors: Hai-Rong
authors: HR
authors: Lu
authors: Mei-Gang
authors: MG
authors: Gu
authors: Qiang
authors: Q
authors: Huang
authors: Jin-jiang
authors: JJ
authors: Huang
authors: Wan-Ying
authors: WY
authors: Lu
authors: Hong
authors: H
authors: Lu
authors: Qing-Shan
authors: QS
authors: Huang
mesh_terms: D003903:Deuterium / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D008248:Lysostaphin / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D012965:Sodium Chloride / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism
chemical_list: D012965:Sodium Chloride; D006859:Hydrogen; D003903:Deuterium; D008248:Lysostaphin
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