title: Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin.
abstract: VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARAtwo-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSAsensory component has not hitherto been identified. Here we employ purified, intact and active VanSAmembrane 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.
authors.affiliation: School of Pharmacy &Biomedical Sciences, Membranes, Membrane Proteins &Peptides Research Group, University of Central Lancashire, Preston, PR1 2HE, United Kingdom.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Phillips-Jones
authors.affiliation: National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD United Kingdom.
authors.forename: Guy
authors.initials: G
authors.lastname: Channell
authors.affiliation: School of Pharmacy &Biomedical Sciences, Membranes, Membrane Proteins &Peptides Research Group, University of Central Lancashire, Preston, PR1 2HE, United Kingdom.
authors.forename: Claire J
authors.initials: CJ
authors.lastname: Kelsall
authors.affiliation: School of Pharmacy &Biomedical Sciences, Membranes, Membrane Proteins &Peptides Research Group, University of Central Lancashire, Preston, PR1 2HE, United Kingdom.|Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.
authors.forename: Charlotte S
authors.initials: CS
authors.lastname: Hughes
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: Simon G
authors.initials: SG
authors.lastname: Patching
authors.affiliation: National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD United Kingdom.
authors.forename: Vlad
authors.initials: V
authors.lastname: Dinu
authors.affiliation: National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD United Kingdom.|School of Health Sciences, University of Nottingham, Nottingham, NG7 2HA United Kingdom.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Gillis
authors.affiliation: National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD United Kingdom.|School of Health Sciences, University of Nottingham, Nottingham, NG7 2HA United Kingdom.
authors.forename: Gary G
authors.initials: GG
authors.lastname: Adams
authors.affiliation: National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD United Kingdom.
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Harding
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002942:Circular Dichroism; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D016983:Enterococcus / Q000201:enzymology*; D000071677:Histidine Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D057446:Hydrodynamics; D013058:Mass Spectrometry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D012996:Solutions; D014461:Ultracentrifugation; D014640:Vancomycin / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D014640:Vancomycin; D000071677:Histidine Kinase