Structure-based functional characterization of repressor of toxin (Rot), a central regulator of Staphylococcus aureus virulence

Abstract

Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure of Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that R(91), at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y(66), predicted to bind within the major groove, contributes to Rot interaction with target promoters. Evaluation of Rot binding to different activated and repressed promoters revealed that certain mutations on Rot exhibit promoter-specific effects, suggesting for the first time that Rot differentially interacts with target promoters. This work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.

FIG 1

Rot structure. (A) The Rot dimer is shown in a ribbon representation from a side view. Monomers are colored in either cyan or green. The chloride ion is represented as a magenta sphere. (B) The sequence of the region in the crystal structure from residues 6 to 133 plus three residues from the 6×His tag is shown with the secondary structure elements, including five α-helices and a two-stranded β-sheet. The structural motifs are color coded: the dimerization core helices (α1, α2, and α5) in green, the helix-turn-helix (HTH) (α3 and α4) containing the recognition helix (RH) (α4) in magenta, and the wing (β1 and β2) in dark blue. The underlined residues make dimerization contacts. Red residues are predicted to make contact with protein partners, purple residues are predicted to make nonspecific contact with DNA, and orange residues are predicted to make specific contact with DNA. (C) Structural motifs listed in panel B are shown on the Rot monomer. (D) The Rot dimer is shown in a B-factor putty representation where the thickness of a region is proportional to its local B-factor and thus its flexibility.

FIG 2

Hydrophobic dimerization interface and hydrogen bonds in the Rot dimer. (A) Surfaces of Rot molecules. The two monomers in the Rot dimer are colored cyan and green, respectively, and the hydrophobic residues are depicted in yellow. (B) Water-mediated contacts span the Rot dimerization interface. The side chains of residues that extend across the dimerization interface are colored in magenta. The key water molecules that participate in dimerization are shown as small red spheres. (C) Closeup view of panel B in which hydrogen bonds are shown as black dotted lines. (D) Closeup view of the hydrogen bonds formed between the side chains of the symmetry-related Q¹²⁴ from each Rot monomer.

FIG 3

Surface charge distribution and sequence conservation among SarA family members. (A) Electrostatic profiles of SarA family proteins. The electrostatic surface potentials of each protein are colored by charge, with blue representing positive and red representing negative charge. Proteins are oriented with their WHTH facing forward. Proteins are depicted from most to least acidic isoelectric point (left to right, top to bottom). (B) Sequence alignment of WHTH domains of SarA family members. The height of each letter in the top portion of the figure represents the prevalence of that amino acid at the particular position. (C) Sequence conservation of SarA family members projected onto Rot surface. Each residue on the surface of Rot is colored according to sequence conservation, with blue representing the most and red the least conserved. Sequence conservation was defined by alignment generated by BLAST. The figure was generated by the ConSurf program (53).

FIG 4

Prediction of domains important for Rot function. (A) Model of Rot-DNA interaction. Rot dimers (cyan and green) are depicted as ribbons, while the DNA (brown) backbones and bases are depicted as thin tubes with an overall transparent surface. The side chains of Y⁶⁶ and R⁹¹ are shown. (B) Zoomed-in view of modeled interaction at major and minor grooves. The side chains are shown for Y⁶⁶ in the RH with the major groove of DNA and R⁹¹ of the wing with the minor groove. (C) Optimal Docking Area (ODA) analysis. ODA was used to predict interfaces for protein-protein interactions (44). Red spheres indicate locations on the surface of Rot where protein interactions are likely to occur. (D) Side chains are shown for residues predicted from the ODA analysis to interact with protein partners.

FIG 5

Targeted Rot residues for site-directed mutagenesis. (A) Residues altered to generate mutant Rot proteins are shown in stick representation. Orange residues are predicted to make specific contacts with DNA. Purple residues are predicted to make nonspecific contacts with DNA. Red residues are predicted to be involved in interactions with protein partners. Distances between the Y⁶⁶ residues and the R⁹¹ residues in the two monomers are reported. (B) The WHTH region is shown in a closeup view with the side chains of selected residues in stick representation. The chloride ion is shown as a red sphere. (C and D) The HTH region is shown from two orientations. (E) Closeup view of the region predicted by ODA to be involved in interaction with protein partners.

FIG 6

Mutant Rot proteins display functional defects. Mutant rot alleles carried on plasmid pOS1Plgt were transformed into an S. aureus reporter strain containing the sGFP gene under the control of the ssl7 promoter, which is activated by Rot. Left, Pssl7 activation by Rot containing group amino acid mutations in regions predicted to be important for Rot-mediated regulation. Right, Pssl7 activation by Rot proteins containing single amino acid mutations selected from group substitution mutants. Activation activity of Rot mutants was assessed by GFP fluorescence in cultures grown to post-exponential phase. Values are averages of results from three independent experiments ± standard deviations.

FIG 7

Mutations in Rot differentially affect activation, repression, and DNA binding. (A to D) Transcript levels of ssl7 (A) and spa (B), which are activated by Rot, or lukE (C) and hla (D), which are repressed by Rot, were quantified by qRT-PCR from strains containing the wild-type Rot or its mutant proteins. Transcript levels were quantified in units of relative quantitation (RQ) and compared to that of the empty vector (Neg) or the wild-type Rot protein (WT). Data bars represent the average of results from 3 experiments ± standard deviation. (E and F) EMSA of purified WT and mutant Rot proteins incubated with either the ssl7 (E) or lukED (F) promoter containing a biotin tag. Promoter DNA probes alone (Neg) or preincubated with Rot-His proteins were separated by PAGE. DNA probes were visualized using streptavidin DyLight. “P” denotes unbound DNA probe. “S” denotes shifted band resulting from Rot-DNA complex. The asterisk denotes a nonspecific band.