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 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.

Molecular mechanisms of vancomycin resistance

“…Experimental evidence for this model has been provided in a study that used biophysical approaches such as analytical ultracentrifugation and near-UV circular dichroism (CD) to monitor the changes in oligomerization and conformation of detergent-solubilized VanS A in the presence of vancomycin. 51 Another study further supported this model demonstrating that vancomycin induced significant thermostability and conformational changes in purified VanS A, while other potential signals such as the peptidoglycan components Ala-D-γ-Glu-Lys-D-Ala-D-Ala, N-acetylmuramic acid or D-Ala-D-Ala had no such effect. 52 However, other studies contested this model, reporting the lack of a VanS A -vancomycin complex even at high concentrations of vancomycin and suggesting that the enzymatic activities of detergent or amphipol-solubilized VanS A are not altered in the presence of vancomycin.…”

“…Recognition of vancomycin would trigger VanS A dimerization, autophosphorylation, and transfer of the phosphoryl group to VanR A . Experimental evidence for this model has been provided in a study that used biophysical approaches such as analytical ultracentrifugation and near‐UV circular dichroism (CD) to monitor the changes in oligomerization and conformation of detergent‐solubilized VanS A in the presence of vancomycin . Another study further supported this model demonstrating that vancomycin induced significant thermostability and conformational changes in purified VanS A, while other potential signals such as the peptidoglycan components Ala‐ d ‐γ‐Glu‐Lys‐ d ‐Ala‐ d ‐Ala, N ‐acetylmuramic acid or d ‐Ala‐ d ‐Ala had no such effect .…”

The antibiotic vancomycin induces complexation and aggregation of gastrointestinal and submaxillary mucins

“…However, it should also be borne in mind that, for most glycopeptide antibiotics studied to date [72][73][74] including vancomycin 66 , it has been shown that binding to depsipeptide targets is accompanied by formation of asymmetric, back-to-back homodimers of the glycopeptide in aqueous solution and that this is mediated by sugar-sugar recognition 75,76 . Although glycopeptide antibiotics are known to bind or interact with protein targets such as d-alanyl-d-alanine and VanS 4,6 , precipitation with another glycoprotein, heparin, has also been reported previously. However, this only appears to occur at relatively high concentrations of both components such as those used in intravenous lines (vancomycin at 1-5 mg/mL and heparin at 1-1000 Units/mL) [77][78][79][80] .…”

“…Analysis of the variation of M w with loading concentration revealed dissociation constants in the range 25-75 µM, commensurate with a relatively weak association. That study 4 , alongside companion studies 6,7 , also demonstrated a weak interaction with the A-type bacterial VanS histidine protein kinase involved in the activation of vancomycin resistance, at least in aqueous solution.…”

Tapping into Synchrotron and Benchtop Circular Dichroism Spectroscopy for Expanding Studies of Complex Polysaccharides and their Interactions in Anoxic Archaeological Wood

“…Circular dichroism (CD) (and synchrotron circular dichroism (SCD)) spectroscopy is widely used to study chiral molecules, in particular biomolecules in solution and in thin amorphous dry films. It is often used to elucidate secondary structural composition and to investigate tertiary structural conformational changes in biological monomeric, oligomeric and polymeric molecules including proteins and peptides (e.g., [1][2][3][4][5][6][7][8][9][10]), nucleic acids (e.g., [4][5][6]11]), carbohydrates and polysaccharides (e.g., [4,[12][13][14][15][16][17][18][19][20]). In the definition of CD, it is a chiroptical method that requires the molecule under study to possess optical activity (chirality).…”