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

DOI: 10.1038/srep46180 DOI Help

Authors: Mary K. Phillips-jones (University of Central Lancashire) , Guy Channell (University of Nottingham) , Claire J. Kelsall (University of Central Lancashire) , Charlotte S. Hughes (Diamond Light Source; University of Central Lancashire) , Alison E. Ashcroft (University of Leeds) , Simon G. Patching (University of Leeds) , Vlad Dinu (University of Nottingham) , Richard B. Gillis (University of Nottingham) , Gary G. Adams (University of Nottingham) , Stephen E. Harding (University of Nottingham)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Scientific Reports , VOL 7

State: Published (Approved)
Published: April 2017
Diamond Proposal Number(s): 8516

Open Access Open Access

Abstract: VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA 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.

Journal Keywords: Biochemical assays; Membrane proteins

Subject Areas: Biology and Bio-materials


Instruments: B23-Circular Dichroism

Documents:
srep46180.pdf