X‐ray structure of an M. jannaschii DNA‐binding protein: Implications for antibiotic resistance in S. aureus

Abstract

Bacterial resistance to commonly used antibiotics, such as the β-lactams and vancomycin, has become a major public health concern.1, 2 Research in the area of methicillin resistance revealed a number of auxiliary genes that augment the function of PBP2a, the gene product primarily responsible for methicillin resistance in Staphylococcus aureus.3 Although some of these auxiliary methicillin resistance proteins contribute to well-characterized biosynthetic pathways (e.g., cell wall metabolism), functions for several of these gene products remain elusive.4 This article describes the X-ray structure of a Methanococcus jannaschii protein that is related to one such auxiliary gene product, RUSA223 [Genbank Code: CAB60749]. The S. aureus auxiliary gene product RUSA223 and its ortholog from M. jannaschii [Genbank Code: NP_248571, PSI-BLAST E-value = 10−25, sequence identity = 23%] were expressed in Escherichia coli BL21(DE3) with N-terminal His-tags. Following Ni-affinity chromatography, each protein was purified to homogeneity using an S Sepharose ion exchange column. Initial crystallization trials with the S. aureus protein yielded neither crystals nor promising leads. Dynamic light scattering studies demonstrated that RUSA223 was severely aggregated at submilligram concentrations in aqueous solution. Mj223 yielded crystals in a number of related conditions. Diffraction-quality crystals grown via hanging drop vapor diffusion against a solution containing 0.1 M Tris HCl 8.0, 30% PEG8000, and 10% dioxane belong to the hexagonal space group P65 (unit cell: a = 57.0 Å, c = 208.4 Å; two protomers per asymmetric unit). Selenomethionine-labeled Mj223, prepared as described above, yielded crystals under very similar conditions. Following cryoprotection with added glycerol (25% v/v), X-ray data were collected at two wavelengths in the vicinity of the Se K-edge from a single crystal maintained at 100 K. Diffraction data were processed with DENZO/SCALEPACK5 (Table I), and the heavy atom substructure (four selenium sites/asymmetric unit) was elucidated with SnB.6 Se atomic positions were refined, and experimental phases were calculated with MLPHARE7 at 2.8 Å resolution (figure of merit = 0.64). Density modification yielded an interpretable electron density, which allowed tracing of the entire polypeptide chain (Met1-Asp151). This initial model was subjected to refinement with CNS8 against diffraction data obtained from an S-Met crystal, giving a final R-factor of 26.2% with R-free = 29.3% (Table I). The N-terminal portion of Mj223 resembles a helix-turn-helix (HTH) winged-helix DNA binding motif,9 which suggests that it functions as a transcription factor. The C-terminal region of the protein is composed of two leucine-rich α-helices, which form a dimer with a neighboring protomer that is related by noncrystallographic two-fold symmetry [Fig. 1(a)]. Dynamic light scattering studies documented that the protein is indeed a dimer in solution (data not shown). The order of secondary structural elements in Mj223 is H1−H2−S1−H3-H4−S2−S3−H5−H6 (where H and S denote α-helices and β-strands, respectively). α-helices H5-H6 and H5′-H6′ (′ denotes the second protomer) dimerize to form an antiparallel four-helix bundle (buried surface area = 4551 Å2), which represents a novel dimerization motif not previously observed in dimeric transcription factors. Mj223 homodimer structure: (a) ribbon representation of the Mj223 dimer with labeled secondary structure elements, colored blue (α-helices) and orange (β-strands) in one protomer with the second protomer colored red; (b) ribbon representation of Mj223 docked with linear B-form DNA; (c) GRASP15 representation of the chemical properties of the solvent-accessible surface of Mj223, calculated using a water probe radius of 1.4 Å. Solvent-accessible surface has been color-coded for the calculated electrostatic potential (red and blue, respectively, represent electrostatic potentials <−8 and >+8 KBT, where KB is the Boltzmann constant and T is temperature). Calculations were performed with an ionic strength of zero and dielectric constants of 80 and 2 for solvent and protein, respectively. The surface of the winged-helix domain is both basic and hydrophobic. The winged-helix domain is one of the most common DNA-binding motifs responsible for controlling transcription initiation in both bacteria and eukaryotes.9 We believe that the Mj223 dimer binds to a symmetric pair of half-sites by presenting α-helices H4 and H4′ to the major groove of duplex B-form DNA [Fig. 1(b)]. The calculated electrostatic potential for the putative DNA-binding surface is both basic and hydrophobic, and the separation of the two recognition α-helices is compatible with major groove binding [Fig. 1(c)]. The linear extent of the predicted DNA footprint is about 40 Å, which corresponds to two adjacent 7 base pair (bp) recognition sites related by two-fold symmetry [Fig. 1(b)]. Mj223 is structurally similar to the eubacterial