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

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 HapR_V2) 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 HapR_V2 to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapR_V2 (HapR_V2G), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR _V2 and HapR_V2G 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.