Phosphorylation-Induced Activation of the Response Regulator VraR from Staphylococcus aureus: Insights from Hydrogen Exchange Mass Spectrometry Yu-Hong Liu 1 , Antoaneta Belcheva 2 , Lars Konermann 1 ⁎ and Dasantila Golemi-Kotra 2,3 ⁎ 1 Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7 2 Departments of Biology and Chemistry, York University, Toronto, Ontario, Canada M3J 1P3 3 Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC 27402-6170, USA Received 1 May 2009; received in revised form 26 May 2009; accepted 4 June 2009 Available online 9 June 2009 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 spectro- metry 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 α8–turn–α9 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 establish- ment of noncovalent protein–protein interactions and intradomain contacts at the expense of previously existing interdomain bonds. α9 appears to be packed against the receiver domain in nonactivated VraR. Support is presented for α1 as a dimerization interface in phosphorylated VraR, whereas protein–protein interactions for nonphosphorylated VraR are impeded by extensive disorder in this region. © 2009 Elsevier Ltd. All rights reserved. Edited by C. R. Matthews Keywords: two-component system; signal transduction; antibiotic resistance; MRSA/VRSA; conformational dynamics Introduction Signal transduction across biological membranes allows cells to sense and respond to changes in their environment. In bacteria, most of these transduction processes are carried out by two-component sys- tems that consist of a membrane-bound sensor histidine kinase (HK) and a cytoplasmic response regulator (RR). As a result of an external stimulus, HK undergoes phosphorylation at a histidine residue. The phosphate is then transferred to an aspartate on a cognate RR. Most RRs share a similar two-domain structure, with a highly conserved N- *Corresponding authors. E-mail addresses: konerman@uwo.ca; d_golemi@uncg.edu. Abbreviations used: VraR, vancomycin-resistance- associated regulator; HK, histidine kinase; RR, response regulator; MRSA, methicillin-resistant Staphylococcus aureus; VRSA, vancomycin-resistant MRSA; VraR-P, phosphorylated VraR; HDX, hydrogen/deuterium exchange; MS, mass spectrometry. doi:10.1016/j.jmb.2009.06.017 J. Mol. Biol. (2009) 391, 149–163 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.