Structural genomics of Caenorhabditis elegans: Triosephosphate isomerase

Introduction. Triosephosphate isomerase (TIM, E.C. 5.3.1.1) catalyzes the reversible isomerization of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate in the glycolytic pathway. It is a well-studied enzyme conserved in function across eukarya, bacteria, and archaea. The “TIM barrel” represents one of the most common protein folds and has been found in variety of proteins with different functions. All known TIM structures comprise approximately 250 amino acid residues per monomer and function as homodimers. It has been proposed that the isomerization reaction proceeds through an enediolate intermediate formed by substrate deprotonation. Crystal structures of TIM and its complexes with transition-state analogs have revealed conformational changes associated with substrate binding and a glutamate residue conveniently positioned to abstract and transfer a proton from one carbon of the substrate to another. The crystal structure of TIM from Caenorhabditis elegans (ceTIM) presented here was solved as a part of the structural genomics project on the C. elegans genome with 19,099 predicted genes. We employed a recently described phasing approach based on derivatization with halides, which seems to be applicable for high-throughput crystallographic projects. Both crystallographically independent ceTIM molecules have been found in the closed conformation with one sulfate and one acetate ion in the substratebinding site. The sidechain of the catalytic residue Glu164 has been refined in a dual conformation, which is relevant for the mechanism of proton transfer. Experimental. The protein expression in Escherichia coli and purification were performed as reported previously (also see http://www.sgce.cbse.uab.edu). As a result of Gateway cloning, the protein was produced with a hexahistidine tag and an eight-amino-acid peptide at both the N-terminus and the C-terminus. Initial crystallization conditions were obtained with the screening kit WIZARD I (Emerald BioStructures) and also with NATRIX (Hampton Research). Diffraction-grade crystals were grown at 22°C by vapor diffusion in hanging drops consisting of 3 L protein solution and 3 L well solution (2 M ammonium sulfate and 50 mM sodium acetate buffer, pH 5.5). The protein stock solution at 16 mg/mL was in 15 mM sodium N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), pH 7.5. The crystals are monoclinic, space group P21, a 36.40 Å, b 64.37 Å, c 105.71 Å, 91.5°, and the asymmetric unit contains two protein molecules. One such crystal was soaked for 10 min in a mother liquor with 0.5 M sodium iodide, dipped in mother liquor with 25% (v/v) glycerol, and flash-frozen in liquid nitrogen. Anomalous data to 2-Å resolution were collected at 170°C on an Raxis IV using the CuK radiation generated by a rotating anode (Table I). No attempts were made to collect the Bijvoet pairs close in time. The data were processed in Denzo/Scalepack, and the structure was solved by the single-wavelength anomolous diffraction (SAD) method in SOLVE. The anomalous Patterson map revealed 12 iodide sites with partial occupancies that were used for protein phasing. After density modification in RESOLVE, the refined phases provided a quality map suitable for automatic model building of approximately 75% of the amino acid residues by RESOLVE. A native diffraction data set was collected to 1.7 Å at the Stanford Synchrotron Radiation Laboratory (SSRL) beam line 9-1, with the wavelength at 0.976 Å. The native data were processed in HKL2000 and used for further structure refinement in Crystallography & NMR System (CNS). The final model [Protein Data Bank (PDB) code 1MO0] consists of two protein chains with a total of 502 amino acid residues, 461 water molecules, 5 sulfate ions, and 2 acetate ions. The crystallographic R-factor is 18.3%, and R-free is 21.3%, with no cutoffs. The model was validated in MolProbity and meets standards for the high-resolution structures. Results and Discussion. Soaking with iodide proved to be a quick and robust phasing method that can even use data collected on an in-house X-ray source. The refinement against native data resulted in defined protein chains without breaks in the 2Fo-Fc electron density map at 1.1 level. However, the Nand C-terminal peptides from the expression vector were just partially resolved in chain A