Evidence on how a conserved glycine in the hinge region of HapR regulates its DNA binding ability: Lessons from a natural variant

Mitesh Dongre, Naorem Santa Singh, Chetna Dureja, Nagesh Peddada, Ashish K. Solanki, Ashish, Saumya Raychaudhuri

Research output: Contribution to journalArticlepeer-review

Abstract

HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued anunderstanding of anobservationofanatural protease-deficient non-O1, non-O139 variant V. choleraestrain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapRV2) harboring a substitutionof glycine to aspartate at position39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapRV2 to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapRV2 (HapRV2G), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR V2 and HapRV2G proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins,weacquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a "Y" shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.

Original languageEnglish (US)
Pages (from-to)15043-15049
Number of pages7
JournalJournal of Biological Chemistry
Volume286
Issue number17
DOIs
StatePublished - Apr 29 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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