Crystal structure of the Vibrio cholerae quorum-sensing regulatory protein HapR

Abstract

Quorum sensing in Vibrio cholerae involves signaling between two-component sensor protein kinases and the response regulator LuxO to control the expression of the master regulator HapR. HapR, in turn, plays a central role in regulating a number of important processes, such as virulence gene expression and biofilm formation. We have determined the crystal structure of HapR to 2.2-A resolution. Its structure reveals a dimeric, two-domain molecule with an all-helical structure that is strongly conserved with members of the TetR family of transcriptional regulators. The N-terminal DNA-binding domain contains a helix-turn-helix DNA-binding motif and alteration of certain residues in this domain completely abolishes the ability of HapR to bind to DNA, alleviating repression of both virulence gene expression and biofilm formation. The C-terminal dimerization domain contains a unique solvent accessible tunnel connected to an amphipathic cavity, which by analogy with other TetR regulators, may serve as a binding pocket for an as-yet-unidentified ligand.

FIG. 1.

Structure of HapR. (A) HapR monomer viewed from the side. The DNA binding surface is at the bottom in this orientation. Helices are rainbow-colored according to the secondary structure succession from blue for the N-terminal helix α1 to red for C-terminal helix α9. Secondary structural elements are labeled accordingly. Helix α3 residues T53 and F55, which have been mutated in the present study, are shown in magenta. The N and C termini are indicated. (B) Side view of the HapR dimer, with one chain in magenta and one chain colored as in panel A. (C) View of the final HapR model showing 2.9-Å electron density at 1.0σ from the initial, experimentally phased, and solvent-flattened electron density map obtained directly from RESOLVE (49). (D) The same view of the final HapR model showing 2.2-Å electron density from a composite omit map calculated with CNS (3) using the final model, contoured at 1.5σ. The structural figures in the present study were rendered by using the program PYMOL (6).

FIG. 2.

Multiple sequence alignment of TetR family members and secondary structure assignment for HapR based on its crystal structure. The HapR secondary structure is shown above the sequence. Residue numbering is to the right for all proteins and also above the secondary structure elements for HapR. The sequences were aligned by using CLUSTAL W (4) and formatted using ESPript (15). Residues identical to HapR are red, and residues identical in two or more family members are blue. The black boxes show the residues altered in the present study to alanine. Conserved residues in the DNA-binding domain discussed in the text are highlighted in yellow, and residues in the putative binding pocket (shown in Fig. 3C) are in highlighted in cyan. (Accession numbers are shown to the right of each line.) Vc, V. cholerae; Vh, V. harveyi; Mt, M. tuberculosis; Sc, S. coelicolor; Sa, S. aureus; Ec, E. coli.

FIG. 3.

View of the solvent accessible tunnel and putative ligand-binding pocket of HapR. (A) Ribbon diagram of the HapR dimer, oriented as in Fig. 1B, showing the location of the tunnel and cavity in blue, monomer A in magenta, and monomer B in green. The surfaces in this figure were generated by using the program SURFNET (28) and rendered by using PYMOL (6). (B) Top view of the tunnel and cavities looking down the HapR dimer interface. (C) View of residues containing atoms surrounding the solvent isolated pocket in monomer B. (D) Electrostatic surface potential of the HapR dimer, oriented as in panel A. Blue and red indicate positive and negative charges, respectively. The black arrow points to the electropositive entrance to the solvent accessible tunnel and pocket, visible slightly to the right of the center of the dimer. The electrostatic surface was calculated by using the APBS (2) plug-in to PYMOL (6). Atomic charges and radii were calculated by using the AMBER force field option at the online PDB2PQR service (8).

FIG. 4.

Comparison of HapR with DNA-bound apo QacR and dequalinium-bound QacR. (A) SSM (27) produced superposition of DNA-bound apo QacR (PDB 1JT0, chain B) shown in cyan and dequalinium-bound QacR (1JT6:A) shown in green. For clarity, only helices α5, α6, and α7 (amino acids 76 to 136) and the DNA-binding domain (amino acids 2 to 4) are shown. Note that in the dequalinium-bound QacR structure, helix α5 is extended by a full turn, causing the DNA-binding domain to move in relation to helices α5 to α7, as indicated by the arrow. (B) Alignment of HapR, shown in orange, and QacR-dequalinium, shown in green, showing that helix α5 is extended in both of these structures to a similar degree and that the DNA-binding domains do not align. The pronounced kink in helix α7 of HapR is indicated. C, Alignment of HapR, in orange, and apo QacR-DNA, in cyan, showing the close alignment of the DNA-binding domains.

FIG. 5.

Model of the HapR-DNA complex. A side view of the model of the HapR dimer (one chain in cyan and the other in green) bound to DNA is shown. The primary DNA interactions are predicted to be via helix α3 (yellow), which fit into adjacent major grooves on the same face of the DNA. The model was made by aligning the HapR dimer with the QacR dimer-DNA structure (PDB code 1JT0, chains B and D).

FIG. 6.

Mutational analysis of residues in HapR helix α3. (A) Binding of wild-type and mutant HapR proteins to the aphA promoter. Lane 1, no protein added; lanes 2 to 5, wild-type HapR; lanes 6 to 9, HapR T53A; lanes 10 to 13, HapR F55A. The first lane in each set has 5 nM (2.5 ng) protein, the second lane has 50 nM (25 ng), the third lane has 200 nM (100 ng), and the fourth lane has 500 nM (250 ng). (B) Influence of HapR mutations on the expression of an aphA-lacZ promoter fusion in V. cholerae. Strains were grown under AKI conditions (19) at 37°C for 3.5 h. From left to right are shown GK178 (wt), KSK1815 (ΔhapR), WL521 (T53A), and WL522 (F55A). (C) Western analysis of crude extracts (17.5 μg) of the strains in panel B using anti-HapR antibody.