title: Phosphorylation-induced activation of the response regulator VraR from Staphylococcus aureus: insights from hydrogen exchange mass spectrometry.
abstract: A two-component system consisting of the histidine kinase vancomycin-resistance-associated sensor and the response regulator vancomycin-resistance-associated regulator (VraR) allows Staphylococcus aureus to sense antibiotic-related cell wall stress and to mount a suitable response. An experimental structure of full-length VraR is not available yet, but previous work points to similarities between VraR and the well-characterized NarL. This work employs hydrogen exchange mass spectrometry to gain insights into the phosphorylation-induced activation of VraR, a process that primes the protein for dimerization and DNA binding. Whereas VraR is highly dynamic, phosphorylated VraR shows less extensive deuteration. This rigidification is most dramatic within the receiver domain, which carries the phosphorylation site D55. Alterations in the DNA-binding domain are much less pronounced. Changes in deuteration within the receiver domain are consistent with a Y-T coupling mechanism. In analogy to NarL, the activation of VraR is thought to involve separation and subsequent reorientation of the two domains, thereby allowing the alpha8-turn-alpha9 element to engage in DNA binding. The current work suggests that this structural transition is triggered by a reduction in the effective length of the linker through enhanced hydrogen bonding. In addition, separation of the two domains may be favored by the establishment of noncovalent protein-protein interactions and intradomain contacts at the expense of previously existing interdomain bonds. alpha9 appears to be packed against the receiver domain in nonactivated VraR. Support is presented for alpha1 as a dimerization interface in phosphorylated VraR, whereas protein-protein interactions for nonphosphorylated VraR are impeded by extensive disorder in this region.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Yu-Hong
authors.initials: YH
authors.lastname: Liu
authors.forename: Antoaneta
authors.initials: A
authors.lastname: Belcheva
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
authors.forename: Dasantila
authors.initials: D
authors.lastname: Golemi-Kotra
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C478170:VraR protein, Staphylococcus aureus