Crystal structure of a single‐stranded DNA‐binding protein (TM0604) from Thermotoga maritima at 2.60 Å resolution (2006)

The TM0604 gene of Thermotoga maritima encodes a single-stranded DNA-binding protein (SSB) with a molecular weight of 16,166 Da (residues 1–141) and a calculated isoelectric point of 4.8. SSB plays essential roles in DNA replication, recombination, and repair.1-3 SSB, also known as helix-destabilizing protein, binds tightly to single-stranded DNA (ssDNA) as a homotetramer. SSB from E. coli can bind long ssDNAs in two main binding modes, which occlude 35 or 65 nucleotides per tetramer, depending on how many SSB subunits from the tetramer interact with ssDNA. In the SSB35 binding mode, two SSB subunits per tetramer interact with ssDNA and self-assemble into long clusters. In the SSB65 binding mode, all four SSB subunits interact with ssDNA and do not form clusters.4 Closely related variants of SSB are encoded in the genome of a variety of large, self-transmissible plasmids. The eukaryotic mitochondrial proteins that bind ssDNA are structurally and evolutionarily related to prokaryotic SSB and are probably involved in mitochondrial DNA replication.5 Here, we report the crystal structure of TM0604, which was determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).6 Protein Production and Crystallization. SSB from Thermotoga maritima (TIGR: TM0604, Swiss-Prot: Q9WZ73) was amplified by PCR from genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH2T7, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth using the Escherichia coli strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze/thaw procedure in Lysis Buffer [50 mM Tris pH 7.9, 50 mM NaCl, 10 mM imidazole, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-chelating resin (GE Heathcare) pre-equilibrated with Lysis Buffer. The resin was washed with Wash Buffer [50 mM potassium phosphate pH 7.8, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP], and the target protein was eluted with Elution Buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. The eluate was buffer exchanged with Buffer Q [20 mM Tris pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl and applied to a RESOURCE Q column (GE Heathcare) pre-equilibrated with the same buffer. The target protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q. The appropriate RESOURCE Q fractions were pooled and further purified using a Superdex 200 column (GE Healthcare) with elution in Crystallization Buffer [20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP]. The appropriate Superdex 200 fractions were pooled and concentrated for crystallization assays to 10 mg/mL by centrifugal ultrafiltration (Millipore). Molecular weight and oligomeric state of the target protein were determined using a 1.0 × 30 cm Superdex 200 column (GE Heathcare) in combination with static light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 8.0, 150 mM NaCl. The protein was crystallized using the nanodroplet vapor diffusion method7 with standard JCSG crystallization protocols.6 The crystallization reagent contained 25% MPD, 0.1M Tris pH 8.0. An additional 10% MPD (35% final concentration) was added as a cryoprotectant. The crystals were indexed in the orthorhombic space group F222 (Table I). Native diffraction data were collected to 2.3 Å resolution at the Advanced Light Source (ALS, Berkeley, CA) on beamline 5.0.3. The dataset was collected at 100 K using an ADSC Q4 CCD detector. Data were integrated and reduced using Mosflm8 and then scaled with the program SCALA from the CCP4 suite.9 Data statistics are summarized in Table I. Diffraction was anisotropic, resulting in incomplete data in the higher resolution shells. Intensity falloff is greatest in the b* axis direction. Because the data are incomplete to 2.3 Å, we define the nominal resolution as 2.6 Å, which is the resolution of a dataset that is 100% complete and has the same number of reflections as observed in the current dataset.10 There are 739 observed reflections between 2.6 and 2.3 Å (39% complete for this shell). The structure was determined with the JCSG molecular replacement pipeline11 using E. coli SSB protein (PDB: 1sru, sequence identity 34%) as the search model. Refinement was carried out using RESOLVE,12 REFMAC5,13 and XFIT.14 Refinement statistics are summarized in Table I. Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool,15 MolProbity,16 SFcheck 4.0,9 and WHATIF 5.0.17 Figure 1(B) was adapted from an analysis using PDBsum,18 and all others were prepared with PyMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of TM0604 have been deposited within the PDB and are accessible under the code 1z9f. Crystal structure of SSB from Thermotoga maritima. (A) Stereo ribbon diagram of TM0604 monomer color-coded from N-terminus (blue) to C-terminus (red). The N-terminus corresponding to the actual start of SSB is labeled. The α-helix H1 and β-strands (β1–β6) are labeled. Disordered regions are indicated by dashed lines. (B) Diagram showing the secondary structural elements of TM0604 superimposed on its primary sequence. The α-helix, β-strands, β-turns, and disordered regions, with corresponding sequence in brackets, are indicated. The three-dimensional structure of TM0604 [Fig. 1(A)] was determined to a nominal resolution of 2.60 Å by the molecular replacement (MR) method using the coordinates of the E. coli SSB protein (PDB: 1sru, sequence identity of 34%)19 as the search model. Data collection, model, and refinement statistics are summarized in Table I. The final model contains only 88 of the 141 residues and includes one monomer (residues 1–23, 26–37, 49–85, and 93–108), one residue from the His-tag, and 14 water molecules. No electron density was observed for residues 24 and 25, 38 to 48, 86 to 92, 109 to 141, and the remaining residues of the expression and purification tag. The Matthews' coefficient (Vm)20 is 1.94 Å3/Da, and the estimated solvent content is 36.2%. The Ramachandran plot, produced by MolProbity,16 shows that 93.8%, 97.5%, and 2.5% of the residues are in favored, allowed, and disallowed regions, respectively. The outlier residues are Met1 (ϕ = 172.4° and ψ = −57.8°) and Ser2 (ϕ = 104.7° and ψ = 19.9°), which are located at the N-terminus, but have good electron density. The TM0604 monomer contains six β-strands (β1–β6) and one α-helix (H1) [Fig. 1(A,B)], with about 37% of the structure being disordered. The total β-strand and α-helical content is 34.1% and 8.4%, respectively. The Structural Classification of Proteins database (SCOP) classifies this protein as an oligonucleotide-binding fold (OB-fold).21 The OB-fold comprises a closed, five-stranded β-barrel architecture (β1, β3–β6) that packs, in the case of TM0604, against one α-helix (H1) and an additional β-strand (β2) [Fig. 1(A)]. Analytical size exclusion chromatography in combination with static light scattering indicates the oligomeric state to be a tetramer. A tetramer is also consistent with the analysis of crystallographic packing using the PQS server,22 as well as what has been reported for SSB from E. coli (PDB: 1eyg) [Fig. 2(A,B)]. (A) Stereo ribbon diagram of a superposition of the TM0604 tetramer (blue) and an SSB-ssDNA complex from E. coli (gray). (B) Stereo view of a model of a TM0604-ssDNA complex in surface representation with ssDNA depicted as sticks. The model is based on the SSB-ssDNA complex from E. coli. The surface is colored according to electrostatic potential in a range where red is negative (−84 kT/e) and blue is positive (+ 84 kT/e). A structural similarity search, performed with the coordinates of TM0604 using the DALI23 server, reveals the structure of SSB from Mycobacterium tuberculosis (PDB: 1ue1),24 with an RMSD of 1.3 Å over 86 aligned Cα atoms and a sequence identity of 30%. A superposition of TM0604 with SSB from E. coli (PDB: 1sru)19 reveals an RMSD of 1.5 Å over 79 aligned Cα atoms, whereas an alignment with an SSB-ssDNA complex from E. coli (PDB: 1eyg)4 gives an RMSD of 1.6 Å over 79 aligned Cα atoms, with a sequence identity of 34% in both cases. A structural comparison between tetramers of TM0604 and the SSB-ssDNA complex from E. coli reveals a similar tetrameric architecture and a dramatic decrease of disorder in the loops between 23 to 26, 37 to 49, and 85 to 93 upon binding to ssDNA25 [Fig. 2(A)]. From a number of residues implicated in critical interactions with ssDNA in E. coli SSB (Arg3, Gly15, Trp40, Trp54, Phe60, Lys62 Tyr70, Lys73, Trp88, Met109, and Met111), only Phe60, Tyr70, Lys73, Trp88, and Met109 are strictly conserved in TM0604. Nevertheless, a superposition of TM0604 with the SSB-ssDNA complex shows good agreement of surface topography and electrostatic potential and suggests a very similar ssDNA binding mode for TM0604 [Fig. 2(B)]. Currently, the SSB protein family contains more than 400 sequence homologues mainly of bacterial, eukaryotic, and viral origin. Models for TM0604 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM0604. The TM0604 structure reported here represents an SSB protein, whose structure has been determined by X-ray crystallography. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights regarding the role of this protein in DNA replication, recombination, and repair. This work was supported by NIH Protein Structure Initiative grants P50-GM 62411 and U54 GM074898 from the National Institute of General Medical Sciences (http://www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the Advanced Light Source (ALS). The SSRL is a national user facility operated by Stanford University on behalf of the U. S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U. S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.