Crystal structure of a conserved hypothetical protein TT1751 from <i>Thermus thermophilus</i> HB8

Abstract

Thermus thermophilus HB8, an extremely thermophilic bacterium, optimally grows at 70°C.1 Thus, the proteins from this bacterial strain are quite stable at room temperature, easy to purify, and suitable for X-ray crystallographic studies. TT1751 is annotated as a conserved hypothetical protein from T. thermophilus HB8. The TT1751 open reading frame (ORF) encodes a polypeptide chain of 129 amino acids, with a molecular mass of 14.1 kDa. A BLAST analysis of the TT1751 sequence against the nonredundant protein database identified a total of 14 homologues. Proteins with sequence similarities to TT1751 ranging from 28% to 57% exist in archaea and prokaryotes, including the pathogenic bacteria Vibrio vulnificus and Legionella pneumophila (Fig. 1). In the Pfam database, these proteins belong to a Domain of Unknown Function, DUF302,4 which is equivalent to COG3439 in the National Center for Biotechnology Information Database of Clusters of Orthologous Groups.6 All of these proteins were annotated as hypothetical proteins, and their average length is approximately 135 amino acid residues. We now report the crystal structure of TT1751 at 2.0 Å resolution, which is the first structure of a member of the DUF302 protein group. Multiple alignment of conserved regions of the TT1751 superfamily, constructed by using BLAST2 followed by CLUSTALW.3 Strictly conserved and similar residues are represented within a red box and by a red letter, respectively. Vibri, Vibrio vulnificus CMCP6 (gi: 27367302); Legio, Legionella pneumophila (gi: 19881007); Strep, Streptomyces coelicolor A3(2) (gi: 21218722); Magne, Magnetococcus sp. MC-1 (gi: 22999309); Rhodo, Rhodospirillum rubrum (gi: 22965578); Metha, Methanosarcina acetivorans C2A (gi: 20092743); Coryn, Corynebacterium efficiens YS-314 (gi: 25026614); Brady, Bradyrhizobium japonicum (gi: 27380888); Geoba, Geobacter metallireducens (gi: 23054701); Thrmv, Thermoplasma volcanium (gi: 13541839), Halob, Halobacterium sp. NRC-1 (gi: 15789460); Pyroc, Pyrococcus furiosus DSM 3638 (gi: 18977076); Thrma, Thermoplasma acidophilum (gi: 16081642); Ferro, Ferroplasma acidarmanus (gi: 22406850). The figure was generated with ESPrint 2.0.5 Cloning, expression, and purification: The TT1751 gene from T. thermophilus was amplified by polymerase chain reaction (PCR) and subcloned into the pET11b vector. A selenomethionine derivative of the protein was expressed in E. coli B834 (DE3), induced by isopropyl-β-D-thiogalactopyranoside (IPTG). The cell lysate was incubated at 70°C for 30 min and was then centrifuged to remove the denatured protein. The soluble fraction was applied to a Q Sepharose column (Amersham Biosciences) previously equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 50 mM NaCl and 1 mM DTT. The proteins were eluted by using a linear gradient of 0.05 to 1 M NaCl. The fractions containing TT1751 were collected and dialyzed against 20 mM Tris-HCl buffer (pH 8.5) containing 1mM DTT. The solution was applied to a Mono Q HR 5/5 column (Amersham Biosciences) previously equilibrated with 20 mM Tris-HCl buffer (pH 8.5) containing 1 mM DTT. The proteins were eluted by using a linear gradient of 0 to 1 M NaCl. The fractions containing TT1751 were collected and dialyzed against 50 mM HEPES-NaOH buffer (pH 7.0) containing 150 mM NaCl and 1 mM DTTT. The eluted proteins were applied to a Superdex 75 HR 10/30 gel filtration column (Amersham Biosciences), equilibrated with 50 mM HEPES-NaOH buffer (pH 7.0) containing 150 mM NaCl and 1 mM DTT. The purifed protein was concentrated to 6.0 mg/ml using a Centricon filter (Millipore). The yield of the SeMet-substituted TT1751 was 8.1 mg from 6.9 g of wet cells. The protein concentration was measured with a Protein Assay Rapid Kit (Wako). The N-terminal amino acid sequence of the purified TT1751 protein was confirmed with a Procise amino acid sequencer (Applied Biosystems). Crystallization and data collection: The initial crystallization conditions were screened by microb