Crystal structure of TT1662 from Thermus thermophilus HB8: A member of the α/β hydrolase fold enzymes (2005)

As a target for structural genomics, Thermus thermophilus HB8 is a good choice because of its small gene size. The genome sequencing for T. thermophilus is currently being completed. These sequencing efforts have provided us with coding information for thousands of new proteins. Of these proteins, the TT1662 protein has been selected to determine its biological function based on a three-dimensional (3D) structure. The TT1662 protein is 238 amino acids long, with a molecular mass of 26.0 kDa. A sequence homology search by BLAST1 for the TT1662 protein revealed 25% sequence identity to its counterpart in Yersinia pestis strain 91001. Furthermore, Pfam analysis2 predicted that the TT1662 protein could be a phospholipase/carboxylesterase. These results imply that the TT1662 protein is a putative α/β hydrolase. The α/β hydrolases, which are among the most investigated categories of enzymes,3, 4 hydrolyze a variety of substrates, although their fundamental folding manner is conserved.3, 4 As a potential new member of the α/β hydrolase superfamily, the 3D structure of the TT1662 protein has been determined by the multiple-wavelength anomalous dispersion method (MAD) at 1.6 Å. The TT1662 gene was amplified by polymerase chain reaction (PCR) from T. thermophilus HB8 genomic DNA. The PCR product was cloned into the pET11a expression vector (Novagen). The selenomethionine (SeMet)-labeled TT1662 protein was expressed in the Escherichia coli methionine auxotroph B834(DE3). The cells were cultured at 37°C in LeMaster medium5 containing SeMet. After disruption of the cells by sonication, the lysate was incubated at 70°C for 30 min and centrifuged to remove the denatured protein. The supernatant was applied to an SP-Sepharose column equilibrated with 20 mM 2-morpholinoethanesulfonic acid (MES), pH 6.5, 25 mM NaCl (buffer A). The TT1662 protein was eluted by a linear gradient of NaCl to 1 M. Peak fractions were pooled and dialyzed against buffer A. The sample solution was then loaded on a Mono S 5/5 column and eluted. After gel filtration on Superdex75, the protein was concentrated to a final concentration of 4.5 mg/mL by a Centricon filter unit (Amicon). Crystals of the TT1662 protein were obtained in 3 days by the hanging drop vapor diffusion method at 20°C in 20% polyethylene glycol (PEG3350), 0.2 M potassium fluoride (KF). X-ray diffraction data were collected with a MAR charge-coupled device (CCD) detector installed on the BL44B2 at the SPring-8 synchrotron facility (Harima, Japan) using flash-frozen crystals at 100 K. All X-ray diffraction data were integrated and scaled using the HKL2000 package.6 Data collection statistics are summarized in Table I. The crystal structure of the SeMet-labeled TT1662 protein was determined by the MAD method. SOLVE7 was used to locate the selenium sites and to calculate the phases. The initial trial revealed 16 selenium sites. Including known site information, further searches for unknown sites were conducted with SOLVE. A total of 22 selenium sites were finally included for phase calculation. Electron density modification was subsequently performed with the program RESOLVE.7 The automatic tracing procedure by ARP/wARP8 and manual model building by O9 were used to complete the modeling of the protein molecules. The structure was refined using the Crystallography & NMR System (CNS) program package.10 All refinement steps were monitored with the free R-factor, based on 10% of the X-ray data. Following a simulated annealing protocol, the structure was refined using atom-positional and temperature factor refinement, as well as manual model building. The stereochemical quality of the final model was assessed by PROCHECK11 and WHATIF.12 The Ramachandran plot demonstrated that 99.5% of all residues lie in the allowed region. The refinement statistics are presented in Table I. Atomic coordinates have been deposited at the Protein Data Bank (PDB), with the access code 1UFO. The crystal structure of the TT1662 protein shows a canonical α/β hydrolase fold,3 which possesses 8 β-strands (β1 to β8) and 6 α-helices (αA to αF) [Fig. 1(A)]. These β-strands form a predominantly parallel β-sheet aligned in the order of β1-β2-β4-β3-β5-β6-β7-β8. The β-sheet composes a hydrophobic core flanked by α-helices. As a strong implication of dimerization, the last β-strand (β8) forms an antiparallel β-sheet with the β8 of the neighboring molecule [Fig. 1(A)]. In addition, intermolecular hydrophobic interactions were observed among amino acid residues Leu222, Val226, Ala229, and Phe230 on αFs, and salt bridges were detected between Glu236 and Arg225′ (the prime indicates the neighboring molecule). These interactions contribute to the construction of a homodimer. In the asymmetric unit of the crystal, 3 dimers are included independently. At the dimer–dimer contact surface, many salt bridges and water-mediated hydrogen bonds were found. Structure of the TT1662 protein. All figures were produced with Molscript13 and Raster3D.14 (A) Ribbon diagram of the crystal structure of the TT1662 protein dimer. The black line between the 2 molecules indicates noncrystallographic 2-fold symmetry. (B) 3D arrangement of the catalytic triad. Relevant amino acid residues are depicted in ball-and-stick models. Hydrogen bonds are shown as dotted lines. (C) Superimposed structures of the TT1662 protein (blue) and the carboxylestelase from P. fluorescens (green). For the enzymatic activity, the hydrolase superfamily has the catalytic triad, which is composed of a nucleophile (Ser, Cys, or Asp), an acidic residue, and an absolutely conserved histidine.4 These amino acids correspond to Ser113, Asp183, and His217 for the TT1662 protein. The nucleophile is positioned on the nucleophile elbow, as identified by the consensus sequence Sm-X-Nu-X-Sm (Sm = small residue, X = any residue, and Nu = nucleophile),3 corresponding to Gly(111)-Gly-Ser-Leu-Gly(115) in the TT1662 protein. The architecture of the 3 catalytic residues, Ser-His-Asp, is that of the classical triad in serine proteases15 [Fig. 1(B)]. A structural homology search was conducted with DALI server.16 The top homologue of the TT1662 protein is a carboxylesterase from Pseudomonas fluorescens (1AUO),17 with a Z-score of 21.7. Of the 40 structures with high Z-scores (> 10), 39 are hydrolases. Figure 1(C) shows the superimposed structures of the TT1662 protein and the carboxylesterase from P. fluorescens. The overall structures are well superimposed, with an root-mean-square deviation (RMSD) value of 2.4 Å for 191 common Cα-carbons, although β1 is missing in the structure of the carboxylesterase from P. fluorescens. Significant differences were observed at the C-terminal ends of β4 and β6. These moieties are responsible for substrate binding. These structural variations may exist in order to accommodate different substrates.4 We thank Drs. Hiroto Yamaguchi and Takaaki Hikima for their help in data collection at RIKEN beamline BL44B2 of SPring-8, and Naomi Obayashi and Yuki Kamewari for technical assistance with protein production and purification.