Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for...

VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanS_AR_A two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanS_A sensory component has not hitherto been identified. Here we employ purified, intact and active VanS_A membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~(12 ± 2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s - either a ligand induced dimerization and/or compaction of the monomer are considered.