Structure of Plasmodium falciparum Triose-phosphate Isomerase-2-Phosphoglycerate Complex at 1.1-Å Resolution

Sampathkumar Parthasarathy,Kandiah Eaazhisai,Hemalatha Balaram,Padmanabhan Balaram,Mathur R.N Murthy

doi:10.1074/jbc.m308525200

Abstract

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.

Highlights

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-Å resolution
Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have been determined at atomic resolution, shows that certain interactions are common to the three structures, 2-phosphoglycerate is neither a substrate nor a transition state analogue
Triose-phosphate isomerase (TIM)1 is a ubiquitous glycolytic enzyme that catalyzes the isomerization of dihydroxy acetone phosphate (DHAP) and glyceraldehyde 3-phosphate through the intermediate formation of cis-enediol(ate(s))

Summary

EXPERIMENTAL PROCEDURES

Crystallization and Data Collection—Cloning, overexpression, and purification of PfTIM followed the procedures described earlier [18, 19]. 2PG as the sodium salt was purchased from FLUKA chemicals. Crystallization and Data Collection—Cloning, overexpression, and purification of PfTIM followed the procedures described earlier [18, 19]. The reservoir solution contained 8 –24% polyethylene glycol 1450 and 100 mM sodium acetate (pH 4 –5.0). Protein and the ligand were mixed well, in molar ratios of either 1:50 or 1:100 before crystallization and allowed to equilibrate for 1 h. The crystal belongs to the monoclinic P21 space group with a dimer in the asymmetric unit (see Table I). Structure Solution and Refinement—The orientation and position of the dimeric molecule in the P21 cell were determined using the molecular replacement program AMoRe [21, 22] starting from the structure of native PfTIM determined at 2.1 Å (PDB code 1YDV) [19] as the phasing model. Visual inspection of 2Fo Ϫ Fc and Fo Ϫ Fc Fourier electron density maps and manual model

Intensity data statistics Radiation wavelength Space group Cell parameters

Mean isotropic equivalent B factor Protein Solvent Ligand atoms

RESULTS

DISCUSSION