Allosteric regulation of the Plasmodium falciparum cysteine protease falcipain-2 by heme.

During the erythrocytic cycle of Plasmodium falciparum malaria parasites break down host hemoglobin, resulting in the release of free heme (ferriprotoporphyrin IX). Heme is a generator of free radicals that cause oxidative stress, but it is detoxified by crystallization into hemozoin inside the food vacuole. We evaluated the interaction of heme and heme analogues with falcipain-2, a P. falciparum food vacuole cysteine protease that plays a key role in hemoglobin digestion. Heme bound to falcipain-2 with a 1:1 stoichiometry, and heme inhibited falcipain-2 activity against both human hemoglobin and chromogenic peptide substrates through a noncompetitive-like mechanism. A series of porphyrin analogues was screened for inhibition of falcipain-2, demonstrating a minor contribution of iron to heme-falcipain-2 interaction, and revealing dependence on both propionic and vinyl groups for inhibition of falcipain-2 by heme. Docking and molecular dynamics simulation unveiled a novel, inducible heme-binding moiety in falcipain-2 adjacent to the catalytic site. Kinetic data suggested that the noncompetitive-like inhibition was substrate inhibition induced by heme. Collectively these data suggest that binding of heme to falcipain-2 may limit the accumulation of free heme in the parasite food vacuole, providing a means of heme detoxification in addition to crystallization into hemozoin.

Falcipain-2 inhibition by suramin and suramin analogues.

Falcipain-2 is a cysteine protease of the malaria parasite Plasmodium falciparum that plays a key role in the hydrolysis of hemoglobin, a process that is required by intraerythrocytic parasites to obtain amino acids. In this work we show that the polysulfonated napthylurea suramin is capable of binding to falcipain-2, inhibiting its catalytic activity at nanomolar concentrations against both synthetic substrates and the natural substrate hemoglobin. Kinetic measurements suggest that the inhibition occurs through an noncompetitive allosteric mechanism, eliciting substrate inhibition. Smaller suramin analogues and those with substituted methyl groups also showed inhibition within the nanomolar range. Our results identify the suramin family as a potential starting point for the design of falcipain-2 inhibitor antimalarials that act through a novel inhibition mechanism.

Allosteric activation of human α-thrombin through exosite 2 by suramin analogs.

Thrombin is a serine protease that plays fundamental roles in hemostasis. We have recently elucidated the crystal structure of thrombin in complex with suramin, evidencing the interaction through the anion binding exosite 2. Here, we show that the activity of thrombin toward natural and synthetic substrates is enhanced by suramin as well as analogs of suramin at a low micromolar range prior to an inhibitory component at higher concentrations. Suramin analogs substituted by phenyl and chlorine instead of methyl were the most efficient in promoting allosteric activation, with an enhancement of enzymatic activity of 250% and 630% respectively. We discuss the importance of exosite 2 as a regulatory site for ligands in both the procoagulant and inhibitory scenarios.

Structural and thermodynamic analysis of thrombin:suramin interaction in solution and crystal phases.

Suramin is a hexasulfonated naphthylurea which has been recently characterized as a non-competitive inhibitor of human alpha-thrombin activity over fibrinogen, although its binding site and mode of interaction with the enzyme remain elusive. Here, we determined two X-ray structure of the thrombin:suramin complex, refined at 2.4 A resolution. While a single thrombin:suramin complex was found in the asymmetric unit cell of the crystal, some of the crystallographic contacts with symmetrically related molecules are mediated by both the enzyme and the ligand. Molecular dynamics simulations with the 1:1 complex demonstrate a large rearrangement of suramin in the complex, but with the protein scaffold and the more extensive protein-ligand regions keep unchanged. Small-angle X-ray scattering measurements at high micromolar concentration demonstrate a suramin-induced dimerization of the enzyme. These data indicating a dissimilar binding mode in the monomeric and oligomeric states, with a monomeric, 1:1 complex to be more likely to exist at the thrombin physiological, nanomolar concentration range. Collectively, close understanding on the structural basis for interaction is given which might establish a basis for design of suramin analogues targeting thrombin.