Evidence on How a Conserved Glycine in the Hinge Region of HapR Regulates Its DNA Binding Ability

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 an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapR V2 ) harboring a substitution of glycine to aspartate at position 39 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, we acquired 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.Studies on the quorum-sensing signal network of Vibrio cholerae have produced a rich harvest of data where the periodic appearance and performance of two regulatory proteins, namely LuxO and HapR, determine the fate of a plethora of disparate cellular events (1). Of these, HapR has been given the status of a master regulator because it controls a wide range of diverse physiological activities, thus exerting a profound influence on the survival and pathogenic potential of this bacterium. Collectively, it represses biofilm development and the production of primary virulence factors (2) while it stimulates the production of HA/protease (3), promotes chitin-induced competence (4), increases resistance to protozoan grazing (5), enhances the survival against oxidative stress (6), and controls the expression of the gene encoding Hcp (7). In a recent effort, Zhu and co-workers have elegantly characterized additional novel direct targets of HapR and illustrated two distinct binding motifs (motif 1 and motif 2) in all target promoters (8). Because it modulates a multitude of diverse cellular parameters, the absence of a functional HapR affects the physiology of V. cholerae to a great extent....

Substitution of Glutamate Residue by Lysine in the Dimerization Domain Affects DNA Binding Ability of HapR by Inducing Structural Deformity in the DNA Binding Domain

“…HapR V2G and HapR V2G -E 117 K proteins were purified by Ni 2+ -Nitrilotriacetic acid chromatography as described previously [6]. Both wild type and variant HapR were cloned into Nde I- BamH I site of the pET15b vector (Novagen) to generate N-terminal 6X His-HapR fusion proteins.…”

“…After induction with 0.4 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG), HapR proteins were purified through Qiagen Ni 2+ -nitrilotriacetic acid columns and subjected to overnight dialysis in a solution of buffer A containing 10 mM Tris-HCl, pH 7.9, 300 mM KCl, 0.1 mM EDTA. Gel mobility shift assay was done essentially as described earlier [6]. Briefly, three fragments of 399, 665 and 467 bp corresponding to promoter regions of aphA , hapA and vc0900 respectively, were amplified with primer pairs as listed in Table 2.…”

“…Further, structural analysis reveals the mutation driven shape change in the DNA binding domains, thereby explaining the loss in DNA binding ability of this HapR variant. In short, molecular analysis of HapR V2 (G 39 D) natural variant illustrates the importance of a conserved glycine residue in the performance of HapR [6] which was not evaluated in the previous studies [4].…”

“…Though many crucial facts about the structural and functional aspects of wild type HapR are available, additional insights into the functionality of this molecule can further be explored by studying natural variants of the same. For instance, a natural non functional HapR variant harboring aspartate in place of glycine residue at position 39 (G 39 D; named as HapR V2 ) clearly elucidates the contributory role of glycine 39 in DNA binding activity of HapR [6]. Further, structural analysis reveals the mutation driven shape change in the DNA binding domains, thereby explaining the loss in DNA binding ability of this HapR variant.…”

“…A flurry of research articles evidences the preponderance of natural variants of quorum sensing regulatory proteins from various strains of Vibrio cholerae [6]–[10]. Of these, strains having non-functional HapR variants are more frequently obtained in nature.…”