Crystal structure of the conserved hypothetical protein TTHA1606 from Thermus thermophilus HB8
Year: 2009
Type: article
Abstract: NIF3 family proteins are highly conserved from bacteria to human [Fig. 1(A)], but their functions remain unclear. NIF3 was originally identified as a NGG1-interacting factor in yeast on two-hybrid screening.1 Several studies of eukaryotic NIF3 homologues suggested that NIF3 family proteins take part in transcription.2, 3 The ybgI gene, the Nif3 family gene of Escherichia coli, is encoded in the operon including endonuclease VII, a DNA repair protein involved in oxidative base damages.4 Microarray analyses indicated that the expression of ybgI was induced by treatment with a DNA damaging agent.5 This suggests a possible function of YbgI in the DNA repair process. (A) Sequence alignment of TTHA1606, E. coli YbgI, and human NIF3LI. Human NIF3LI contains a PII domain in its middle region, which is omitted in this figure. Asterisks indicate conserved residues. (B) The structure of the dimer of TTHA1606 (viewed from the inside of the ring). (C) The structure of the hexamer of TTHA1606 viewed from two sides. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] The crystal structures of E. coli YbgI,6Staphylococcus aureus SA1388,7Streptococcus pneumoniae SP1609, and Bacillus cereus YqfO8 have been determined. In these structures, two metal ions tightly bind in the cavity inside the hexameric troid. Although the coordination of the dinuclear center exhibits some similarity to those of other metal-binding proteins, it remains uncertain how the metal-binding site is involved in the function. The Thermus thermophilus HB8 genome contains the gene encoding the NIF3 family protein TTHA1606, which consists of 242 amino acid residues. TTHA1606 is transcribed constantly9 and translated into a protein under normal growth conditions (unpublished data). In the "Whole-Cell Project," we selected T. thermophilus HB8 for systematic study of the structures and functions of all proteins from a single organism.10 In this research, we determined the structure of TTHA1606. In addition, we found that TTHA1606 has the ability to bind single-stranded DNA (ssDNA). Based on the structure, we discuss the DNA-binding site of TTHA1606. The expression plasmid of THA1606 (RIKEN BioResource Center) was transformed into E. coli BL21(DE3). The transformants were cultured at 37°C to 1 × 108 cell/mL in 1.5 L of a medium containing LB broth supplemented with 50 μg/mL of ampicillin. The cells were then incubated for 5 h in the presence of isopropyl-β-D-thiogalactopyranoside and harvested by centrifugation. Cells (28 g) were lysed by sonication in 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl, and the lysate was incubated at 70°C for 10 min. After centrifugation, TTHA1606 in the supernatant was purified by successive chromatographic steps with Resource ISO, Resource Q, BioScale CHT10-I hydroxyapatite, and HiLoad 16/60 200 pg columns. Purified proteins were stored in 20 mM Tris-HCl (pH 8.0). The crystals of the TTHA1606 protein were obtained under condition number 14 (0.1M HEPES buffer, pH 7.5, containing 0.2M CaCl2) of the Crystal Screen crystal screening kit (Hampton Research) using the 96-well sitting-drop vapor-diffusion method11 at 20°C. A 0.5 μL aliquot of a crystallization reagent, consisting of 28% (w/w) PEG 400 and 0.1M HEPES buffer (pH 7.5), containing 0.2M CaCl2, was mixed with 0.5 μL of a 25 mg/mL protein solution, and then the mixture was covered with 15 μL of silicone and paraffin oil. Data were collected with the RIKEN Structural Genomics Beamline II (BL26B2)12 at SPring-8 (Hyogo, Japan). The data were processed using the HKL-2000 program suite.13 The structure was solved using E. coli YbgI (PDB code 1NMP) as the model for molecular replacement using Molrep.14 The automatic tracing procedure in the program ARP/wARP15 was utilized to build the initial model. Model refinement was carried out using the programs Xtalview16 and CNS.17 According to PROCHECK,18 the final model has 85.2% of the residues in the most favored region of the Ramachandran plot and no residues in the disallowed regions. Data-collection statistics and processed data statistics are presented in Table I. (The coordinates are available in the Protein Data Bank under accession code 2YYB and the nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number DDBJ/EMBL/GenBank AP008226.) A synthesized 37-mer oligonucleotide (5′-ATGTGAATC AGTATGGTTACTATCTGCTGAAGGAAAT-3′) was labeled at the 5′ end with [γ-32P]ATP using T4 polynucleotide kinase. This oligonucleotide was used as ssDNA. Double-stranded DNA (dsDNA) was made by annealing the labeled oligonucleotide to the complementary oligonucleotide. The reaction was carried out at room temperature in 10 μL of 50 mM Tris-HCl and 100 mM KCl, pH 7.5, containing TTHA1606 and ssDNA or dsDNA. The mixture was electrophoresed and the bands in the gel were analyzed. The overall structure of TTHA1606 was determined at 2.6 Å resolution [Fig. 1(B)]. The asymmetric unit contains two TTHA1606 molecules. Each subunit consists of two interlinked α/β domains. Both domains comprise three layers, that is, two α-helix layers sandwiching one β-sheet. This fold is shared by all structurally characterized NIF3 family proteins.6-8 The apparent molecular mass of the purified TTHA1606 was estimated to be ∼180 kDa by gel filtration (data not shown), suggesting TTHA1606 forms a hexamer. Judging from the result of crystallographic analysis, it is possible for TTHA1606 to exist as a hexameric form, as shown in Figure 1(C). The overall shape of the hexamer can be viewed as a double-ring of two trimers: two trimers in face-to-face contact. The results of electrophoretic mobility-shift assay (EMSA) showed that TTHA1606 bound to ssDNA but not dsDNA [Fig. 2(A)]. To our knowledge, this is the first report of the DNA-binding ability of a NIF3 family protein. Since the lysate of E. coli cells overexpressing TTHA1606 was heated at 70°C for 10 min during the purification process, it was unlikely that DNA-binding proteins of E. coli contaminated the purified preparations. It should be noted that the binding affinity varied from one preparation to another. Some factors in the preparations might affect the structural stability or activity of TTHA1606. (A) The results of EMSA. The reaction mixture contained 10 nM ssDNA (the left six lanes) or dsDNA (the right six lanes) and TTHA1606 at 0, 4.5, 9, 18, 36, and 40 μM. (B) Stereoview of the electrostatic potential map of TTHA1606 (viewed from the inside of the ring). Red and blue represent negatively and positively charged regions, respectively. Two cavities are also indicated. Compared with Figure 1(B) and the panel D in this figure, the dimer in this panel is viewed from a slightly different angle to bring the cleft into view. (C) Stereoview of another cavity indicated in panel B. Side chains are represented by sticks. Residue numbers followed by asterisks indicate residues of the other subunit in the dimer. (D) The conserved region of the TTHA1606 dimer. The amino acids are colored according to their conservation; see the color-coding bar. The troidal ring structure of TTHA1606 prompted us to examine the possibility that ssDNA binds to the inside of the ring. Several observations support this hypothesis. First, the inner surface of the TTHA1606 ring is more conserved as to amino acid sequence than the outside. We mapped levels of amino acid conservation to TTHA1606 structure with the ConSurf server.19 The mapping of the dimer unit revealed that the inside of the ring was more conserved than the outside [Fig. 2(D)]. This suggests that the inside of the ring is important for the functional roles of NIF3 family proteins. In addition, the well-conserved residues are concentrated in and near the clefts found in the inner surface of the ring: each cleft is formed by two subunits [Fig. 2(B)]. Other structurally determined NIF3 proteins have metal ions in the cleft.6-8 The conserved region at the interface of the dimer partially overlaps the metal-binding site in the cleft [Fig. 2(D)]. The purified TTHA1606 contained no metal ion, and the addition of metal ions had no apparent effect on the interaction with ssDNA (data not shown). Many DNA-binding proteins are known to bind DNA without metal ion. However, the metal-binding site of NIF3 family proteins is presumed to be important for unassigned functions. Metal ions may contribute to DNA sequence- or length-specific binding. Alternatively, metal ions may affect the binding to mRNA, modified nucleic acids, or polyphosphates. Second, positively charged region exists near the metal-binding site in the cleft [Fig. 2(B)]. This region comprises another cavity on the opposite side of the cleft. This region is formed by residues of both subunits of the dimer [Fig. 2(C)]. The crystal structures of all NIF3 family proteins also have this cavity. It is possible that a negatively charged ligand binds to this cavity. Although many basic residues are found in this region [Fig. 2(C)], most of them are less conserved [Fig. 2(D)]. However, the side wall of the cleft near this cavity is composed of well-conserved residues [Fig. 2(D)]. Interestingly, Godsey et al. reported that a HEPES molecule bound to this cavity in the B. cereus YqfO structure and that this site is large enough to accommodate a nucleotide.8 These observations imply that this cavity also comprises the ligand-binding site. Finally, TTHA1606 bound to ssDNA, but not to dsDNA. This might be due to restriction of the ligand size by the structure of the binding site. The inside diameter of the ring is ∼24 Å, which is large enough for ssDNA to pass through it, but not for dsDNA. B. cereus YqfO and S. aureus SA1388 contain an insert in the middle of the protein, which forms a PII-like domain in their crystal structures.7, 8 This domain forms trimeric lids that cover the openings on either side of the troidal rings. Godsey et al. suggested that some ligand might cause conformational change in these lids.8 It should be also noted that the pattern of conserved residues are the same with or without the lids.
DOI: 10.1002/prot.22397
Institutions: The University of Osaka, SPring-8
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2016106183