THE HISTIDINE KINASE VraS INTERACTS WITH VANCOMYCIN AND AMPICILLIN THROUGH ITS MEMBRANE-ANCHORED N-TERMINAL DOMAIN

Multidrug-resistant Staphylococcus aureus represents a major global health threat, with the VraTSR three-component system playing a central role in resistance to cell-wall active antibiotics, such as β-lactams and glycopeptides. This regulatory system, composed of the membrane histidine kinase VraS, the cytoplasmic response regulator VraR, and the uncharacterized membrane protein VraT, governs the activation of the cell wall stress stimulon. Despite its importance, the molecular signals that trigger VraTSR activation have remained elusive.

In this study, we investigated the interactions between VraTSR components and antibiotics using complementary biochemical and biophysical approaches. Full-length VraS was overexpressed in E. coli, purified in DDM micelles, and analyzed for antibiotic binding using saturation transfer difference (STD) NMR spectroscopy and antibiotic-derived photoprobes. STD NMR experiments showed interaction of vancomycin and ampicillin with VraS, while antibiotics that do not activate the system in vivo failed to bind. Using a truncated construct (VraS-N-terminal-eGFP), we demonstrated that the antibiotic binding site was in the N-terminal membrane-anchored region of VraS. Photo-crosslinking assays using photoreactive derivatives of vancomycin and ampicillin confirmed the formation of covalent adducts with VraS, but not with VraT, indicating that VraS is the primary antibiotic sensor.

To further explore the topology and activation mechanism of the VraS/VraT complex, we engineered lanthanide-binding tag (LBT) fusion constructs and performed luminescence resonance energy transfer (LRET) assays with a fluorescent β-lactam (Bocillin FL). We confirmed terbium (III) binding to LBT-VraS and detected distance-dependent luminescence changes upon Bocillin FL binding, supporting the use of this system to probe spatial arrangements within the complex. Co-expression and co-purification of LBT-tagged VraS and VraT enabled the study of complex formation in membrane extracts.

Altogether, our findings reveal a direct interaction of β-lactams and glycopeptides with VraS and lay the groundwork for future studies aiming to elucidate conformational changes in the VraTSR system. This work provides a toolkit for structural and functional characterization of histidine kinase–mediated antibiotic sensing, with potential implications for the development of novel antimicrobial strategies.