No abstract

Although the molecular mechanism by which chloroquine exerts its effects on the malarial parasite Plasmodium falciparum remains unclear, the drug has previously been found to interact specifically with the glycolytic enzyme lactate dehydrogenase from the parasite. In this study we have determined the crystal structure of the complex between chloroquine and P. falciparum lactate dehydrogenase. The bound chloroquine is clearly seen within the NADH binding pocket of the enzyme, occupying a position similar to that of the adenyl ring of the cofactor. Chloroquine hence competes with NADH for binding to the enzyme, acting as a competitive inhibitor for this critical glycolytic enzyme. Specific interactions between the drug and amino acids unique to the malarial form of the enzyme suggest this binding is selective. Inhibition studies confirm that chloroquine acts as a weak inhibitor of lactate dehydrogenase, with mild selectivity for the parasite enzyme. As chloroquine has been shown to accumulate to millimolar concentrations within the food vacuole in the gut of the parasite, even low levels of inhibition may contribute to the biological efficacy of the drug. The structure of this enzyme-inhibitor complex provides a template from which the quinoline moiety might be modified to develop more efficient inhibitors of the enzyme.Malaria is one of the major diseases of mankind, claiming 3 million lives worldwide annually. Resistance to existing antimalarial drugs is a large and increasing problem. Recently we have determined the first high-resolution structure of an enzyme from the Plasmodium falciparum parasite, the causative agent of malaria. This 1.7-Å structure (1) of the essential glycolytic enzyme P. falciparum lactate dehydrogenase (pfLDH) 1 has revealed a unique cleft adjacent to the active site, ideally suited as a target for the rational design of inhibitors. Another feature of the structure is a significant displacement of the NADH cofactor relative to other forms of LDH, reflecting that the malarial enzyme has a unique mode of association with the cofactor and hence a distinctive NADH binding pocket. These features suggest pfLDH may form an appropriate target for structure-based design of novel antimalarials.Chloroquine (CQ, Fig. 1, left) and related quinoline compounds have been used extensively throughout the world as prophylactics to prevent the development of malaria. Although the mechanism of action of CQ on the parasite is not completely understood, it is thought to interfere with the function of the food vacuole in the mature stages of the erythrocytic parasite (2). CQ is a weak base and accumulates to high concentrations within the acidic food vacuole (3). Within these vacuoles, hemoglobin is degraded by proteases to provide a supply of amino acids for the parasite, but also producing toxic heme moieties as a by-product (hematin; Fig. 1, right). The parasite normally detoxifies hematin by polymerizing the by-product to hemozoin, although the details of this process are unclear. Because CQ is known to bi...

The HIS3+ gene of Saccharomyces cerevisiae was overexpressed in Escherichia coli and the recombinant imidazoleglycerol-phosphate dehydratase (IGPD) purified to homogeneity. Laser-desorption and electrospray m.s. indicated a molecular ion within 2 units of that expected (23833.3) on the basis of the protein sequence, with about half of the polypeptide lacking the N-terminal formylmethionine residue. IGPD initially purified as an apoprotein was catalytically inactive and mainly a trimer of M(r) 70,000. Addition of Mn2+ (but not Mg2+) caused this to assemble to an active (40 units/mg) enzyme (Mn-IGPD) comprising of 24 subunits (M(r) 573,000) and containing 1.35 +/- 0.1 Mn atoms/polypeptide subunit. An enzyme with an identical activity and metal content was also obtained when the fermenter growth medium of recombinant Escherichia coli was supplemented with MnCl2, and IGPD was purified through as Mn-IGPD rather than as the apoenzyme and assembled in vitro. Inhibition by EDTA indicated that the intrinsic Mn2+ was essential for activity. The retention of activity over time after dilution to very low concentrations of enzyme (< 20 nM) indicated that the metal remained in tight association with the protein. A novel continuous assay method was developed to facilitate the kinetic characterization of Mn-IGPD. At pH 7.0, the Km for IGP was 0.10 +/- 0.02 mM and the Ki value for inhibition by 1,2,4-triazole, 0.12 +/- 0.02 mM. In contrast with other reports, thiols had no influence on catalytic activity. The activity of Mn-IGPD varied with enzyme concentration in such a way as to suggest that it dissociates to a less active form at very low concentrations. Significant inhibition by the product, imidazole acetol phosphate, was inferred from the shape of the progress curve. Titration with, the potent competitive inhibitor, 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate indicated that Mn-IGPD contained 0.9 +/- 0.1 catalytic sites/protomer. The activity nearly doubled in the presence of high concentrations of Mn2+; the apparent Ks for stimulation was 20 microM. The basis of this effect was obscure, since there was no corresponding increase in the titre of active sites. Neither was there a discernable shift in the values of Km or Ki (above), although exogenous Mn2+ did reduce the optimum pH for kcat, from 7.2 to 6.8. On the basis of a single site/subunit, the maximum rate of catalytic turnover at 30 degrees C was 32 s-1.

Mucosal addressin cell-adhesion molecule (MAdCAM-1) is a membrane-bound leukocyte receptor regulating both the passage and retention of leukocytes in mucosal tissues. A crystal structure for the two extracellular amino-terminal domains of human MAdCAM-1 has previously been reported, confirming their expected immunoglobulin superfamily topology. In this study, a second crystal structure of this fragment is described. Although the overall structure is similar to that previously reported, one edge strand in the amino-terminal domain is instead located on the opposite sheet. This alters the arrangement and conformation of amino acids in this region that have previously been shown to be crucial for ligand binding. MAdCAM-1 is also seen to form dimers within the crystal lattice, raising the possibility that oligomerization may influence the biological role of this adhesion molecule.

Imidazoleglycerol phosphate dehydratase (IGPD) from Saccharomyces cerevisiae has been crystallized in the presence of a range of divalent cations using the hanging-drop method of vapour diffusion with ammonium sulfate or polyethylene glycol (PEG) 4000 as the precipitants. X-ray precession photographs have established that the crystals formed with ammonium sulfate (form A) belong to the space group F432, with cell parameter a = 177.5/~ and a single subunit in the asymmetric unit. A preliminary data set collected to 6 A resolution on a two-detector San Diego Multiwire area detector has established that the crystals formed with PEG 4000 (form B) belong to either of the special pair of space groups I23 or 12~3, with cell parameter a = 131.0 A,. A self-rotation function has been calculated using these data and indicates that the cell axes show pseudo fourfold symmetry consistent with a dimer in the asymmetric unit in this crystal form. Light-scattering studies indicate that in the presence of Mn 2÷ and a number of other divalent cations IGPD undergoes assembly to a particle of molecular weight approximately 500kDa. Given the subunit molecular weight of 23 kDa together with the symmetry of the crystals it would indicate that the most likely quaternary structure for this enzyme is based on a 24-mer in 432 symmetry.