STRUCTURE NOTE

Structural Genomics of Caenorhabditis elegans:Triosephosphate IsomeraseJindrich Symersky,1 Songlin Li,1 Mike Carson,1 and Ming Luo1,2*1Southeast Collaboratory for Structural Genomics, Center for Biophysical Sciences and Engineering, University of Alabama atBirmingham, Birmingham, Alabama2Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama

Introduction. Triosephosphate isomerase (TIM, E.C.5.3.1.1) catalyzes the reversible isomerization of dihydroxy-acetone phosphate to D-glyceraldehyde 3-phosphate in theglycolytic pathway.1 It is a well-studied enzyme conservedin function across eukarya, bacteria, and archaea. The“TIM barrel” represents one of the most common proteinfolds and has been found in variety of proteins withdifferent functions.2 All known TIM structures compriseapproximately 250 amino acid residues per monomer andfunction as homodimers.3 It has been proposed that theisomerization reaction proceeds through an enediolateintermediate formed by substrate deprotonation.4 Crystalstructures of TIM and its complexes with transition-stateanalogs have revealed conformational changes associatedwith substrate binding and a glutamate residue conve-niently positioned to abstract and transfer a proton fromone carbon of the substrate to another.5,6

The crystal structure of TIM from Caenorhabditis el-egans (ceTIM) presented here was solved as a part of thestructural genomics project on the C. elegans genome with19,099 predicted genes.7 We employed a recently describedphasing approach based on derivatization with halides,which seems to be applicable for high-throughput crystal-lographic projects.8 Both crystallographically independentceTIM molecules have been found in the closed conforma-tion with one sulfate and one acetate ion in the substrate-binding site. The sidechain of the catalytic residue Glu164has been refined in a dual conformation, which is relevantfor the mechanism of proton transfer.

Experimental. The protein expression in Escherichiacoli and purification were performed as reported previ-ously9 (also see http://www.sgce.cbse.uab.edu). As a resultof Gateway cloning,10 the protein was produced with ahexahistidine tag and an eight-amino-acid peptide at boththe N-terminus and the C-terminus. Initial crystallizationconditions were obtained with the screening kit WIZARD I(Emerald BioStructures) and also with NATRIX (Hamp-ton Research). Diffraction-grade crystals were grown at22°C by vapor diffusion in hanging drops consisting of 3 �Lprotein solution and 3 �L well solution (2 M ammoniumsulfate and 50 mM sodium acetate buffer, pH 5.5). Theprotein stock solution at 16 mg/mL was in 15 mM sodiumN-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid

(HEPES), pH 7.5. The crystals are monoclinic, space groupP21, a � 36.40 Å, b � 64.37 Å, c � 105.71 Å, � � 91.5°, andthe asymmetric unit contains two protein molecules.

One such crystal was soaked for 10 min in a motherliquor with 0.5 M sodium iodide, dipped in mother liquorwith 25% (v/v) glycerol, and flash-frozen in liquid nitrogen.Anomalous data to 2-Å resolution were collected at �170°Con an Raxis IV using the CuK� radiation generated by arotating anode (Table I). No attempts were made to collectthe Bijvoet pairs close in time. The data were processed inDenzo/Scalepack,11 and the structure was solved by thesingle-wavelength anomolous diffraction (SAD) method inSOLVE.12 The anomalous Patterson map revealed 12iodide sites with partial occupancies that were used forprotein phasing. After density modification in RESOLVE,12

the refined phases provided a quality map suitable forautomatic model building of approximately 75% of theamino acid residues by RESOLVE. A native diffractiondata set was collected to 1.7 Å at the Stanford SynchrotronRadiation Laboratory (SSRL) beam line 9-1, with thewavelength at 0.976 Å. The native data were processed inHKL200011 and used for further structure refinement inCrystallography & NMR System (CNS).13 The final model[Protein Data Bank (PDB) code 1MO0] consists of twoprotein chains with a total of 502 amino acid residues, 461water molecules, 5 sulfate ions, and 2 acetate ions. Thecrystallographic R-factor is 18.3%, and R-free is 21.3%,with no cutoffs. The model was validated in MolProbity14

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 usedata collected on an in-house X-ray source. The refinementagainst native data resulted in defined protein chainswithout breaks in the 2Fo-Fc electron density map at 1.1�level. However, the N- and C-terminal peptides from theexpression vector were just partially resolved in chain A

*Correspondence to: Ming Luo, Center for Biophysical Sciences andEngineering, University of Alabama at Birmingham, 1025 18th StreetSouth, Birmingham, AL 35294. E-mail: mingluo@uab.edu

Received 12 November 2002; Accepted 19 November 2002

PROTEINS: Structure, Function, and Genetics 51:484–486 (2003)

© 2003 WILEY-LISS, INC.

and completely disordered in chain B. In course of therefinement, both active sites revealed separated electrondensity residuals that were clearly compatible with sulfateand acetate ions acquired from the crystallization. Theacetate ions were positioned with respect for optimalhydrogen bonding, whereas positioning of sulfate ions wasprompted by the shape of electron density residuals. Inaddition, a significant residual in the proximity of Glu164was consistent with a double conformation of the sidechainof Glu164.

The overall structure and active site of ceTIM are, asexpected, very similar to other fully liganded TIMs (Fig. 1).A least-squares fit of C� atoms between the homodimers ofceTIM (PDB code 1MO0) and a complex of chicken TIMwith phosphoglycolohydroxamate15 (PDB code 1TPH) re-sulted in a root-mean-square (RMS) deviation of 0.94 Å.Both ceTIM molecules assume the “closed” conformation,which is most apparent at loop 6 (residues 164–176),which moves by almost 7 Å toward the active site com-pared to the “open” conformation of an unliganded TIM(PDB code 1YPI). It has been shown previously that thesulfate alone can elicit the closed TIM conformation16;however, the sidechain of the catalytic glutamate was notfound in the well-defined “swung-in” conformation ob-served in high-resolution structures of TIM complexeswith transition-state analogs.15,16

Remarkably, in this structure, both sulfate and acetateions are bound in a mode that closely resembles binding oftransition-state analogs, particularly phosphoglycolic acid(not shown) and phosphoglycolohydroxamate [Fig. 1(b)].In both subunits of ceTIM, the sulfate ion occupies thephosphate-binding site, and the acetate simulates thesugar part of the substrate, interacting with active siteresidues Asn10, Lys12, His94, and Glu164. The key resi-due Glu164 has been refined with 75% of the side chain ina nearly swung-in conformation and 25% in a new confor-

Fig. 1. Active site of C. elegans TIM with acetate and sulfate. Threestructurally conserved water molecules are included as red spheres. TheC. elegans structure is shown as thick atoms and bonds colored by atomtype, with key residues labeled. Dashed lines represent hydrogen bonds.Comparison structures are shown in white, with smaller bonds and atomicradii. Top: Comparison to the open conformation; the 1YPI unligandedstructure of chicken TIM. Bottom: Comparison to the closed conforma-tion; the 1TPH structure of chicken TIM with phosphoglycolohydroxamate.

TABLE I. Data Collection and Refinement Statistics

Anomalous Native

Wavelength [A] 1.5418 0.976Crystal Derivative (Nal) nativeResolution (last shell) [A] 25–2.0 (2.07–2.0) 29.6–1.7 (1.76–1.7)Rsym (last shell) [%] 8.9 (23.1) 3.9 (20.6)Completeness (last shell) [%] 95.1 (83.2) 92.7 (86.5)No. of observations 193,940 174,695Unique reflections 61,089 49,994R-factor (R-free)a [%] 18.3 (21.3)No. of nonhydrogen atoms 4295No. of water sites 461Average B-factor [A2] 16.52

Protein 15.3Water 25.46Sulfate 33.8Acetate 24.4

RMSD bond lengths [A] 0.005RMSD bond angles [°] 1.3Ramachandran plot

Most favorable [%] 91.7Disallowed [%] 0

aThe R-free was calculated using a 5% subset of randomly selected reflections.

TRIOSEPHOSPHATE ISOMERASE 485

mation, which has not yet been observed in TIM struc-tures. Superpositions with other TIM structures withtransition state analogs suggest that, whereas the carboxy-late of Glu164 in the swung-in conformation is positionedto abstract a proton from one carbon atom of the substrate,as proposed earlier, the new conformation shown herebrings the carboxyl group of Glu164 within a convenientdistance for the proton readdition to the next carbon atomof the substrate, as required for the isomerization. It hasalso been shown that there is a proton exchange, whichincludes solvent molecules and possibly other groups of theactive site.17,18 Thus, most protons are not transferredfrom one carbon atom to another in a single step. However,the sidechain of Glu164 is observed in conformations thatcan apparently mediate the initial and final steps of theproton transfer between carbon atoms of the substrate.

Acknowledgement. We thank the Structural Genom-ics of Caenorhabditis elegans (SGCE) production group forproviding the protein.

REFERENCES

1. Putman SJ, Coulson AF, Farley IR, Riddleston B, Knowles JR.Specificity and kinetics of triose phosphate isomerase from chickenmuscle. Biochem J 1972;129:301–310.

2. Branden C-I. The TIM barrel—the most frequently occurringfolding motif in proteins. Curr Opin Struct Biol 1991;1:978–983.

3. Casal JI, Ahern TJ, Davenport RC, Petsko GA, Klibanov AM.Subunit interface of triosephosphate isomerase: Site-directedmutagenesis and characterization of the altered enzyme. Biochem-istry 1987;26:1258–1264.

4. Hall A, Knowles JR. The uncatalyzed rates of enolization ofdihydroxyacetone phosphate and of glyceraldehyde 3-phosphatein neutral aqueous solution: The quantitative assessment of theeffectiveness of an enzyme catalyst. Biochemistry 1975;14:4348–4352.

5. Lolis E, Petsko GA. Crystallographic analysis of the complexbetween triosephosphate isomerase and 2-phosphoglycolate at

2.5-Å resolution: Implications for catalysis. Biochemistry 1990;29:6619–6625.

6. Noble EM, Zeelen JP, Wierenga RK. Structures of the “open” and“closed” state of trypanosomal triosephosphate isomerase, asobserved in a new crystal form: Implications for the reactionmechanism. Proteins 1993;16:311–326.

7. Norvell JC, Machalek AZ. Structural genomics programs at theUS National Institute of General Medical Sciences. Nat StructBiol 2000;7(Suppl):931.

8. Dauter Z, Dauter M, Rajashankar, KR. Novel approach to phasingproteins: Derivatization by short cryo-soaking with halides. ActaCrystallogr D 2000;D56:232–237.

9. Li S, Finley J, Liu Z-J, et al. Crystal structure of the cytoskeleton-associated protein (CAP-Gly) domain. J Biol Chem 2002;277:48596–48601.

10. Walhout AJ, Temple GF, Brasch MA, Hartley JL Lorson MA, Vanden Heuvel S, Vidal M. GATEWAY recombinant cloning: Applica-tion to the cloning of large numbers of open reading frames orORFeomes. Methods Enzymol 2000;328:575–592.

11. Otwinowski Z, Minor W. Processing of X-ray diffraction datacollected in oscillation mode. Methods Enzymol 1997;276:307–326.

12. Terwilliger TC, Berendzen J. Automated structure solution forMIR and MAD. Acta Crystallogr D 1999;D55:849–861.

13. Brunger AT, Adams PD, Clore GM, et al. Crystallography & NMRSystem: A new software suite for macromolecular structuredetermination. Acta Crystallogr D 1998;D54:905–921.

14. Richardson D, Richardson J. MolProbity program suite. Durham,NC: Duke University; 2001.

15. Zhang Z, Sugio S, Kornives EA, Liu KD, Knowles JR, Petsko GA,Ringe D. Crystal structure of recombinant chicken triosephos-phate isomerase-phosphoglycolo-hydroxamate complex at 1-Å reso-lution. Biochemistry 1994;2830–2837.

16. Wierenga RK, Noble MEM, Vriend G, Nauche S, Hol WGJ.Refined 1.83 Å structure of trypanosomal triosephosphate isomer-ase crystallized in the presence of 2.4 M ammonium sulfate: Acomparison with the structure of the trypanosomal triosephos-phate isomerase-glycerol-3-phosphate complex. J Mol Biol 1991;220:995–1015.

17. Knowles JR, Albery WJ. Perfection in enzyme catalysis: Theenergetics of triosephosphate isomerase. Accounts Chem Res1977;10:105–111.

18. Rose IA, Fung W-J, Warms JVB. Proton diffusion in the active siteof triosephosphate isomerase. Biochemistry 1990;29:4312–4317.

486 J. SYMERSKY ET AL.