S. Parthasarathy

Structures of Plasmodium falciparum triosephosphate isomerase complexed to substrate analogues: observation of the catalytic loop in the open conformation in the ligand-bound state.

The glycolytic enzymes of Plasmodium falciparum (Pf) are attractive drug targets as the parasites lack a functional tricarboxylic cycle and hence depend heavily on glycolysis for their energy requirements. Structural comparisons between Pf triosephosphate isomerase (PfTIM) and its human homologue have highlighted the important differences between the host and parasite enzymes [Velanker et al. (1997), Structure, 5, 751-761]. Structures of various PfTIM-ligand complexes have been determined in order to gain further insight into the mode of inhibitor binding to the parasite enzyme. Structures of two PfTIM-substrate analogue complexes, those of 3-phosphoglycerate (3PG) and glycerol-3-phosphate (G3P), have been determined and refined at 2.4 A resolution. Both complexes crystallized in the monoclinic space group P2(1), with a molecular dimer in the asymmetric unit. The novel aspect of these structures is the adoption of the loop-open conformation, with the catalytic loop (loop 6, residues 166-176) positioned away from the active site; this loop is known to move by about 7 A towards the active site upon inhibitor binding in other TIMs. The loop-open form in the PfTIM complexes appears to be a consequence of the S96F mutation, which is specific to the enzymes from malarial parasites. Structural comparison with the corresponding complexes of Trypanosoma brucei TIM (TrypTIM) shows that extensive steric clashes may be anticipated between Phe96 and Ile172 in the closed conformation of the catalytic loop, preventing loop closure in PfTIM. Ser73 in PfTIM (Ala in all other known TIMs) appears to provide an anchoring water-mediated hydrogen bond to the ligand, compensating for the loss of a stabilizing hydrogen bond from Gly171 NH in the closed-loop liganded TIM structures.

On the correlation between the main-chain and side-chain atomic displacement parameters (B values) in high-resolution protein structures.

Correlation coefficients (CCs) between the mean atomic displacement parameters (B values) of the main-chain and the side-chain atoms in selected high-resolution protein structures are distributed over a broad range (0.5-1.0). The distribution of CCs is found to be related to the mean difference in B values of Calpha and Cbeta atoms. High CCs are also associated with the frequent occurrence of consecutive Calphaatoms with relatively high B values. The distribution of CCs and its relation to the mean difference between the B values of Calpha and Cbeta atoms shows dependence on the package used for refinement (X-PLOR, PROLSQ or TNT). These observations reflect the differences in the way B-value constraints are handled in these packages. Further differences are discernible in the distributions for proteins refined using the same package. It is likely that these differences are related to the different refinement protocols or weighting schemes followed by investigators. The resolution of these issues is important for evolving correct strategies for the refinement of the atomic displacement parameters in X-ray diffraction studies of proteins. Furthermore, it may be possible to develop refinement-validation tools by observing the features that are invariant in the distribution of atomic displacement parameters.

Cloning, expression, purification and preliminary X-ray crystallographic studies of 2-methylisocitrate lyase from Salmonella typhimurium

In Salmonella typhimurium, propionate is oxidized to pyruvate via the 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30). Methylisocitrate lyase (molecular weight 32 kDa) with a C-terminal polyhistidine affinity tag has been cloned and overexpressed in Escherichia coli and purified and crystallized under different conditions using the hanging-drop vapour-diffusion technique. Crystals belong to the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 63.600, b = 100.670, c = 204.745 A. A complete data set to 2.5 A resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.