Crystal structure of the conserved hypothetical protein TT1380 from Thermus thermophilus HB8 (2004)

The majority of the proteins identified by genome projects are hypothetical proteins. Recently, the structures of these hypothetical proteins have been determined to discover novel protein folds. Additionally, the determined structures sometimes yield clues about protein function. For example, the crystal structure of hypothetical protein TT1466, from the extremely thermophilic bacterium Thermus thermophilus HB8, has been shown to be highly homologous to that of the Coenzyme A (CoA)-binding domain in succinyl-CoA synthetase, despite a sequence identity of only 14%.1 CoA binding by the TT1466 protein was confirmed experimentally. Here, we describe the crystal structure of hypothetical protein TT1380 from T. thermophilus HB8, consisting of 106 amino acid residues. A BLAST database search2 revealed TT1380 homologs with E-values below 0.003 among 20 genera of bacteria, including Deinococcus radiodurans, Bacillus subtilis and Staphylococcus aureus, and one genus of archea. The sequence identities for the homologs range from 31% to 61%. We show the structure of TT1380 with the rare dimeric assembly at a 1.6 Å resolution and discuss its function based on the structural homologies. Selenomethionyl TT1380 was synthesized using the cell-free system, as previously reported.1, 3 The template DNA was pET11b plasmid bearing the TT1380 gene from T. thermophilus HB8 (pET11b–TT1380). The basal buffer (20 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol) was used in all of the following procedures. After termination of the reaction, the solution was dialyzed against basal buffer containing 50 mM NaCl. The dialyzed solution was transferred to a Q-Sepharose column (Amersham Biosciences) and eluted with basal buffer containing 1M NaCl. The target fractions were pooled and diluted with basal buffer containing 50 mM NaCl, and the solution was transferred to a Resource-Q column (Amersham Biosciences), which was eluted with basal buffer containing 1M NaCl. Ammonium sulfate was added to the target fractions up to a 1.2M final concentration, and the solution was then transferred to a Resource-PHE column (Amersham Biosciences) and eluted with basal buffer. The target fractions were transferred to a Superdex75 gel-filtration column (Amersham Biosciences) run with basal buffer containing 150 mM NaCl. The obtained TT1380 solution was dialyzed against Milli-Q water (Millipore) and then concentrated by centrifugation with a Centricon YM-3 filter (Millipore). The yield was 0.38 mg from 36 mL of the reaction solution, and the final concentration was 10.7 mg/mL. Native TT1380 was overexpressed in E. coli BL21 (DE3) cells bearing the pET11b–TT1380 plasmid. The overexpression method has been reported previously.1 The harvested cells were suspended in basal buffer containing 300 mM NaCl. The crude extract was sonicated, incubated at 70°C for 30 min, and centrifuged. The recovered supernatant was subjected to the same chromatography procedures as mentioned above. The yield was 10.0 mg from 2.7 g of wet cells, and the final concentration was 13.7 mg/mL. Crystals of TT1380 were grown at 20°C by the hanging drop method. The mother liquor for selenomethionyl TT1380 consisted of 100 mM sodium cacodylate (pH 7.0), 160 mM zinc acetate and 3% polyethylene glycol (PEG) 8000. The mother liquor for native TT1380 consisted of 100 mM sodium cacodylate (pH 6.0), 90 mM zinc acetate and 18% PEG 8000. The crystals were shown to belong to the space group C2, with cell dimensions a = 81.8 Å, b = 52.0 Å, c = 62.2 Å, α = γ = 90.0°, β = 124.3°, and to contain two molecules per asymmetric unit. The crystals were cryo-cooled to −173°C in the mother liquor with 30% glycerol. All data sets were collected with a CCD detector (Mar) at the synchrotron radiation source of the BL44B2 station in SPring-8, Harima, Japan, and were processed with HKL2000 and SCALEPACK.4 The selenomethionyl TT1380 crystals were used for multiple-wavelength anomalous dispersion (MAD) phasing5 for selenium and zinc. Although the MAD phasing for selenium was unsuccessful, it was achieved with the MAD data sets for zinc. The heavy atom positions were determined with the program SOLVE,6 followed by density modification with the program RESOLVE.7 The initial model at 2.1 Å resolution was used for structural refinement with the native data set at 1.6 Å. The refinement was carried out using the program REFMAC in the CCP4 program suite.8 The model was built with the program TURBO-FRODO.9 The data statistics are given in Table I. The secondary structure was defined using the program PROCHECK.10 The coordinates are available at the Protein Data Bank with the accession code, 1iuj. The monomeric structure of the TT1380 protein is composed of three α-helices (H1–3) and four β-strands (S1–4) (Fig. 1) and has a ferredoxin-like split βαβ-fold with an antiparallel β-sheet [Fig. 2(a)].11 Sedimentation equilibrium measurements by analytical ultracentrifugation showed that the molecular weight of TT1380 is 23,900 (data not shown). Since the expected molecular weight of the dimer was 24,514, we concluded that most of the TT1380 molecules form homodimers in solution. A dimer of TT1380 is found in the asymmetric unit, and it forms an antiparallel β-sheet barrel with a two-fold symmetry axis [Fig. 2(a)]. The C-terminal β-strand S4 associates with S2 in another monomer in an antiparallel manner. Five zinc ions bind to the side chains of Glu60 and Glu67. This zinc cluster is located on the surface of the homodimer [Fig. 2(a)] and contacts that of the other homodimer in the crystal lattice. Two other zinc ions, in subunits A and B, bind to the imidazole rings of His75 at distance of 2.0 and 2.2 Å, respectively [Fig. 2(a)]. A DALI database search12 revealed that the structure of subunit A of the TT1380 protein is highly similar to that of subunit A of ActVA-Orf6 monooxygenase from Streptomyces coelicolor (PDB code 1lq9, z-score, 12.9) and to that of subunit A of a hypothetical protein, At5G22580 from Arabidopsis thaliana (1rjj, 8.6). The sequence identities are 24% for the former and only 14% for the latter. These proteins are the only ones known to share this dimeric form. Since the sequence identities are rather low, this dimeric form might be utilized in many proteins bearing a ferredoxin-like fold. The crystal structures of ActVA-Orf6 monooxygenase from Streptomyces coelicolor and the hypothetical protein At5G22580 from Arabidopsis thaliana were fitted to that of TT1380 by DALI. Regions in the homolog structures that are equivalent to those in TT1380 are underlined. The α-helices and β-strands are indicated with green and red letters, respectively. The secondary structures of TT1380 are represented by green bars for α-helices and red arrows for β-strands. The His75 residue in the sequence of TT1380 and the Tyr51, Asn62, Trp66, Tyr72 and Arg86 residues in the monooxygenase are shown in bold. The residue numbers are listed in a column on the right side of the sequence alignments The five zinc ions of TT1380 that form a cluster are represented in magenta. Zinc ions (blue) bind to the imidazole rings of His75 (cyan). In the overall structure (a), the α-helices and β-strands in subunit A are shown in green and red, respectively. In the diagram of the zinc binding site, acetyl dithranol (shown in salmon), from the complex structure of ActVA-Orf6 monooxygenase bound to its ligand, is superimposed on the structure of TT1380. The Trp56 and Phe62 residues are colored green and yellow, respectively. The orientation is rotated 50° around the horizontal axis and 30° around the vertical axis compared to the view in (a). ActVA-Orf6 monooxygenase catalyzes the oxidation of an aromatic intermediate, 6-deoxydihydrokalafungin, in the actinorhodin biosynthetic pathway.13 The complex structures of ActVA-Orf6 monooxygenase with quinone analogues suggest that five residues may be important for ligand binding: Tyr51, Asn62, Trp66, Tyr72, and Arg86.14 A structural alignment diagram showed that these residues correspond to Val40, Val52, Trp56, Phe62, and His75 of TT1380, respectively [Fig. 1]. When the complex structure of ActVA-Orf6 monooxygenase with acetyl dithranol (PDB code 1n5s) is superimposed on the structure of subunit B of TT1380, the zinc ion bound to His75 becomes superimposed on the acetyl dithranol molecule [Fig. 2(b)]. The conserved residues Trp56 and Phe62 are located near the superimposed ligand. If TT1380 is a monooxygenase, then Trp56, Phe62, and His75 might be important for its activity. We thank Mr. T. Matsu and Drs. T. Hikima and N. Kamiya for supporting our data collection at BL44B2 at SPring-8. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, and the Ministry of Education, Culture, Sports, Science and Technology of Japan.