Protein inhibitors of proteolytic enzymes regulate proteolysis and prevent the pathological effects of excess endogenous or exogenous proteases. Cysteine proteases are a large family of enzymes found throughout the plant and animal kingdoms. Disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer, rheumatoid arthritis, osteoporosis, and emphysema. A family (I42) of cysteine protease inhibitors (http://merops.sanger.ac.uk) was discovered in protozoan parasites and recently found widely distributed in prokaryotes and eukaryotes. We report the 2.2 A crystal structure of the signature member of the I42 family, chagasin, in complex with a cysteine protease. Chagasin has a unique variant of the immunoglobulin fold with homology to human CD8alpha. Interactions of chagasin with a target protease are reminiscent of the cystatin family inhibitors. Protein inhibitors of cysteine proteases may have evolved more than once on nonhomologous scaffolds.

Falcipain-2 (FP2), the major cysteine protease of the human malaria parasite Plasmodium falciparum, is a hemoglobinase and promising drug target. Here we report the crystal structure of FP2 in complex with a protease inhibitor, cystatin. The FP2 structure reveals two previously undescribed cysteine protease structural motifs, designated FP2 nose and FP2arm, in addition to details of the active site that will help focus inhibitor design. Unlike most cysteine proteases, FP2 does not require a prodomain but only the short FP2 nose motif to correctly fold and gain catalytic activity. Our structure and mutagenesis data suggest a molecular basis for this unique mechanism by highlighting the functional role of two Tyr within FP2 nose and a conserved Glu outside this motif. The FP2arm motif is required for hemoglobinase activity. The structure reveals topographic features and a negative charge cluster surrounding FP2 arm that suggest it may serve as an exo-site for hemoglobin binding. Motifs similar to FP2 nose and FP2arm are found only in related plasmodial proteases, suggesting that they confer malariaspecific functions.cysteine protease ͉ falcipain 2 ͉ inhibitor ͉ malaria ͉ x-ray structure A n estimated 500 million people are infected with malaria worldwide, resulting in over a million deaths annually, mainly among children in subSaharan Africa (www.who.int͞healthtopics͞ malaria͞en). There is an urgent need for new antimalarial therapy, because widespread drug resistance has limited the utility of most available treatments (1). Plasmodium falciparum is responsible for nearly all severe illness and death from malaria. During its life cycle within host red blood cells, the protozoan parasite relies on hemoglobin hydrolysis to supply amino acids for protein synthesis and to maintain osmotic stability (2, 3). The proteases involved in hemoglobin hydrolysis have been validated as promising drug targets (2). Both aspartyl proteases (plasmepsins) and cysteine proteases (falcipains) play a role. Inhibition of either protease class is lethal to the parasite, and inhibition of both is synergistic for parasite killing (4-6). Falcipain-2 (FP2), falcipain-2Ј (FP2Ј), and falcipain-3 (FP3) are papain-family (C1) Clan CA cysteine proteases that cleave native or denatured human hemoglobin (7,8). FP2, the most-abundant and best-studied enzyme among the falcipains, is a prime target for drug development. When the FP2 gene was deleted from P. falciparum by homologous recombination, trophozoites accumulated undegraded hemoglobin (9). Moreover, synthetic inhibitors targeting C1 proteases halted P. falciparum growth in cell culture and cured malaria in animal models (6,(10)(11)(12).When compared with family C1 proteases from other organisms, mature falcipains contain two unique sequence motifs: an extension at the N terminus (17 aa for FP2) and an insertion between the catalytic His and Asn near the C terminus (14 aa for FP2). Although the N-terminal extension has been shown to mediate folding of FP2 in the absence of a prodomain (13,14), ...

Falcipain-2 (FP2) is a papain family cysteine protease and important hemoglobinase of erythrocytic Plasmodium falciparum parasites. Inhibitors of FP2 block hemoglobin hydrolysis and parasite development, suggesting that this enzyme is a promising target for antimalarial chemotherapy. FP2 and related plasmodial cysteine proteases have an unusual 14-aa motif near the C terminus of the catalytic domain. Recent solution of the structure of FP2 showed this motif to form a ␤-hairpin that is distant from the enzyme active site and protrudes out from the protein. To evaluate the function of this motif, we compared the activity of the wild-type enzyme with that of a mutant lacking 10 aa of the motif ( ⌬10 FP2). ⌬10 FP2 had nearly identical activity to that of the wild-type enzyme against peptide substrates and the protein substrates casein and gelatin. However, ⌬10 FP2 demonstrated negligible activity against hemoglobin or globin. FP2 that was inhibited with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (FP2 E-64 ) formed a complex with hemoglobin, but ⌬10 FP2 E-64 did not, indicating that the motif mediates binding to hemoglobin independent of the active site. A peptide encoding the motif blocked hemoglobin hydrolysis, but not the hydrolysis of casein. Kinetics for the inhibition of ⌬10 FP2 were very similar to those for FP2 with peptidyl and protein inhibitors, but ⌬10 FP2 was poorly inhibited by the inhibitory prodomain of FP2. Our results indicate that FP2 utilizes an unusual motif for two specific functions, interaction with hemoglobin, its natural substrate, and interaction with the prodomain, its natural inhibitor.M alaria is one of the most important infectious diseases in the world. Plasmodium falciparum, the most virulent human malaria parasite, is believed to cause hundreds of millions of illnesses and over a million deaths each year (1). The control of malaria has been hindered by increasing resistance of malaria parasites to available drugs (2). New antimalarial drugs, ideally directed against new targets, are urgently needed (3). Among potential new targets for antimalarial therapy are proteases that hydrolyze hemoglobin. Intraerythrocytic malaria parasites break down hemoglobin in an acidic food vacuole to supply amino acids for parasite protein synthesis and to maintain osmotic stability (4, 5). Multiple proteases appear to participate in hemoglobin processing (6). Cysteine protease inhibitors block hemoglobin hydrolysis, indicating that cysteine proteases play a key role in this process (7). Falcipain-2 (FP2) and falcipain-3 (FP3) are papain-family cysteine proteases of erythrocytic stages of P. falciparum that localize to the food vacuole and readily hydrolyze hemoglobin (8, 9). Disruption of the FP2 gene led to the accumulation of undegraded hemoglobin in trophozoites, confirming a critical role for this enzyme in hemoglobin hydrolysis (10). Inhibition of FP2 and related proteases led to a block in parasite development (11, 12) and cured mice with murine malaria (13, 14), and efforts to develop...

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The serine protease factor Xa (FXa) is inhibited by ecotin with picomolar affinity. The structure of the tetrameric complex of ecotin variant M84R (M84R) with FXa has been determined to 2.8 A. Substrate directed induced fit of the binding interactions at the S2 and S4 pockets modulates the discrimination of the protease. Specifically, the Tyr at position 99 of FXa changes its conformation with respect to incoming ligand, changing the size of the S2 and S4 pockets. The role of residue 192 in substrate and inhibitor recognition is also examined. Gln 192 from FXa forms a hydrogen bond with the P2 carbonyl group of ecotin. This confirms previous biochemical and structural analyses on thrombin and activated protein C, which suggested that residue 192 may play a more general role in mediating the interactions between coagulation proteases and their inhibitors. The structure of ecotin M84R-FXa (M84R-FXa) also reveals the structure of the Gla domain in the presence of Mg(2+). The first 11 residues of the domain assume a novel conformation and likely represent an intermediate folding state of the domain.

The protease inhibitor ecotin fails to inhibit thrombin despite its broad specificity against serine proteases. A point mutation (M84R) in ecotin results in a 1.5 nM affinity for thrombin, 10(4) times stronger than that of wild-type ecotin. The crystal structure of bovine thrombin is determined in complex with ecotin M84R mutant at 2.5 A resolution. Surface loops surrounding the active site cleft of thrombin have undergone significant structural changes to permit inhibitor binding. Particularly, the insertion loops at residues 60 and 148 in thrombin, which likely mediate the interactions with macromolecules, are displaced when the complex forms. Thrombin and ecotin M84R interact in two distinct surfaces. The loop at residue 99 and the C-terminus of thrombin contact ecotin through mixed polar and nonpolar interactions. The active site of thrombin is filled with eight consecutive amino acids of ecotin and demonstrates thrombin's preference for specific features that are compatible with the thrombin cleavage site: negatively charged-Pro-Val-X-Pro-Arg-hydrophobic-positively charged (P1 Arg is in bold letters). The preference for a Val at P4 is clearly defined. The insertion at residue 60 may further affect substrate binding by moving its adjacent loops that are part of the substrate recognition sites.

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