Crystal structure of SmcR, a quorum-sensing master regulator of Vibrio vulnificus, provides insight into its regulation of transcription

Abstract

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. SmcR, a homologue of Vibrio harveyi LuxR, has been proposed as a quorum-sensing master regulator of Vibrio vulnificus, an opportunistic human pathogen. Previous studies demonstrated that SmcR is essential for the survival and pathogenesis of V. vulnificus, indicating that inhibiting SmcR is an attractive approach to combat infections by the bacteria. Here, we determined the crystal structure of SmcR at 2.1 A resolution. The protein structure reveals a typical TetR superfamily fold consisting of an N-terminal DNA binding domain and a C-terminal dimerization domain. In vivo and in vitro functional analysis of the dimerization domain suggested that dimerization of SmcR is vital for its biological regulatory function. The N-terminal DNA recognition and binding residues were assigned based on the protein structure and the results of in vivo and in vitro mutagenesis experiments. Furthermore, protein-DNA interaction experiments suggested that SmcR may have a sophisticated mechanism that enables the protein to recognize each of its many target operators with different affinities.

FIGURE 1.

Crystal structure of SmcR. a, ribbon representation of SmcR. b, structural comparison of TetR superfamily proteins. SmcR, the V. cholerae transcriptional regulator, HapR (PDB code 2PBX), and the S. aureus multidrug binding transcriptional repressor, QacR (PDB code 1JUP), are displayed in black, green, and magenta, respectively. The structures are rotated 180° about the y axis of a. Unless otherwise noted, figures were prepared using PyMOL.

FIGURE 2.

Dimerization of SmcR and its biological effects. a, C-terminal antiparallel helices of SmcR form a four-helix dimerization motif. The structure is rotated 90° about the y axis of Fig. 1a. Residues at the dimer interface are shown. b, site-directed mutagenesis analysis of the residues critical for dimerization using the bacterial two-hybrid system in E. coli BTH101. The phenotypes were assessed by their cellular β-galactosidase activities using cultures in the stationary growth phase (A₆₀₀ = 2.0–3.0). Expression levels of all of the mutant proteins were compared by Western blot analysis. The black and blue arrows indicate the products of pHS451 and pHS450, respectively. NC indicates negative control as in supplemental Fig. S1 (also see text). WT, wild type. c, effects of the dimerization of SmcR on the transcriptional regulation of vvpE in V. vulnificus. Cellular β-galactosidase activities were measured using cultures in the stationary growth phase (A₆₀₀ = 2.0–3.0). NC indicates negative control (DH0602 containing empty vector pJH0311). d, DNA binding effect of SmcR dimerization and its mutant to the vvpE regulatory region. A 200-bp DNA fragment from the upstream region of vvpE was radioactively labeled and then used as a DNA probe. The radiolabeled fragments (20 ng) were mixed with increasing amounts of wild-type or mutant SmcR (30, 60, 90, and 120 nm in 1st to 4th lanes, respectively), and then resolved on a 4% polyacrylamide gel. B, bound DNA; F, free DNA.

FIGURE 3.

DNA binding domain and DNA recognition of SmcR. a, electrostatic surface potential of the SmcR dimer. The DNA binding domain is rotated 90° about the x axis. The recognition helix of each subunit is indicated. b, superimposition of the DNA binding domains of SmcR and QacR complexed with DNA (PDB code 1JT0). SmcR (molecules A and B) and QacR (molecules A and C) are shown in green and magenta, respectively. c, putative DNA recognition residues. Basic residues predicted to bind with DNA sugar-phosphate backbones are displayed. DNA recognition residues on the helix-turn-helix motif are shown based on the QacR structure.

FIGURE 4.

Structure-related functional sequence conservation between LuxR homologous proteins. Shown above the alignments are elements of the secondary structure of SmcR. The numbering shown is from SmcR. Blue triangles indicate the residues critical for binding with DNA. Open green circles represent residues involved in the formation of the putative ligand-binding pocket. Purple triangles represent residues located at the dimer interface. Biological sources and accession codes for the sequences are as follows: LuxR, V. harveyi (gi:107933356); OpaR, Vibrio parahemeolyticus (gi:28899290); VanT, Vibrio anguillarum (gi:18104604); HapR, V. cholerae (gi:87133250); and LitR, V. fischeri (gi:59712784). Sequence alignments were assembled using T-COFFEE software and visualized using ESPript software, both located on the ExPASy Proteomics Server.

FIGURE 5.

Mutational analysis of putative DNA-binding residues. a, electrophoretic gel mobility shift assay for binding of SmcR mutants to the vvpE regulatory region. A 200-bp DNA fragment from the upstream region of vvpE was radioactively labeled and then used as a DNA probe. The radiolabeled fragments (20 ng) were mixed with wild-type or mutant SmcRs (100, 150, and 200 nm in 1st to 3rd lanes, respectively) and were then resolved on a 4% polyacrylamide gel. N, no protein; WT, wild-type SmcR; Dele, deletion mutant (Asp-2 to Arg-11); B, bound DNA; F, free DNA. b, effect of putative DNA recognition residues on the stability of the helix-turn-helix motif. T_mvalue of each protein is indicated. c, effects of putative DNA-binding residues on the positive transcriptional activation of SmcR in the P_vvpE::lacZ transcriptional fusion system in V. vulnificus DH0602. The phenotypes were assessed by cellular β-galactosidase activities. NO, DH0602 containing pJH0311.

FIGURE 6.

Effects of putative DNA-binding residues on the negative transcriptional repression of SmcR in the P_{VV2_1398}::luxAB transcriptional fusion system in E. coli DH5α. Relative light units were calculated by dividing the luminescence by the A₆₀₀ of each strain cultured as described in the text. NO, DH5α containing empty pBAD24BS vector and pKS0710; WT, wild type.

FIGURE 7.

Isothermal titration calorimetry of SmcR and DNA interactions. Titrations of P_vvpE and P_vvpE-mod against SmcR were performed. Upper panel, raw data obtained from 25 automatic injections of 6-μl aliquots of SmcR against duplex DNA. Lower panel, integration plot of the data calculated from the raw data. Each set of duplex DNA is indicated in the panel. Thermodynamic values are shown in the table.