Crystal structure of the SarS protein from Staphylococcus aureus

Abstract

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). One of these determinants, protein A (spa), is activated by sarS, which encodes a 250-residue DNA-binding protein. Genetic analysis indicated that the agr locus likely mediates spa repression by suppressing the transcription of sarS. Contrary to SarA and SarR, which require homodimer formation for proper function, SarS is unusual within the SarA protein family in that it contains two homologous halves, with each half sharing sequence similarity to SarA and SarR. Here we report the 2.2 A resolution X-ray crystal structure of the SarS protein. SarS has folds similar to those of SarR and, quite plausibly, the native SarA structure. Two typical winged-helix DNA-binding domains are connected by a well-ordered loop. The interactions between the two domains are extensive and conserved. The putative DNA-binding surface is highly positively charged. In contrast, negatively charged patches are located opposite to the DNA-binding surface. Furthermore, sequence alignment and structural comparison revealed that MarR has folds similar to those of SarR and SarS. Members of the MarR protein family have previously been implicated in the negative regulation of an efflux pump involved in multiple antibiotic resistance in many gram-negative species. We propose that MarR also belongs to the winged-helix protein family and has a similar mode of DNA binding as SarR and SarS and possibly the entire SarA protein family member. Based on the structural differences of SarR, SarS, and MarR, we further classified these winged-helix proteins to three subfamilies, SarA, SarS, and MarR. Finally, a possible transcription regulation mechanism is proposed.

FIG. 1.

Overall structure of SarS. (a) The initial MIR map at the beta-hairpin region of S1 with the final SarS model, which is one of the most flexible parts in the entire SarS structure. (b) The final 2Fo-Fc map with the final SarS model at the same region as in panel a. (c) Ribbon diagram of the three-dimensional structure of the SarS protein. The first domain, S1, is shown in green. The second domain, S2, is yellow. (d) Orientation of the panel, 180°. Two hydrophobic cores are labeled. All figures were prepared using RIBBONS (6), except for Fig. 1a and b, which were prepared using BobScript (15).

FIG. 2.

The detailed hydrophobic cores of SarS. (a) The hydrophobic core 1, which brings S1 and S2 tightly together. (b) The hydrophobic core 2, which stabilizes the winged-helix motif.

FIG. 3.

Comparison of the two SarS domains. S1 is shown in green, and S2 is yellow.

FIG. 4.

The electrostatic potential surface of SarS calculated by GRASP (29), with charges of +1 for Lys and Arg, −1 for Glu and Asp, and zero for all other residues. The color bar from red to blue represents the potential from negative to positive, defined as in GRASP. (a) The putative DNA-binding surface; (b) the possible activation motifs.

FIG. 5.

Comparison of SarS with SarR. (a) The overlap of winged-helix motifs from SarS and SarR. (b) The overlap of entire SarS over SarR homodimer. SarS is shown in green, and SarR is blue.

FIG. 6.

Comparison of SarS and MarR. (a) The overlap of winged-helix motifs from SarS and MarR. (b) The overlap of the entire SarS over the MarR homodimer. SarS is shown in green, and SarR is yellow.