The Structure of Chagasin in Complex with a Cysteine Protease Clarifies the Binding Mode and Evolution of an Inhibitor Family

University of California, Berkeley, Berkeley, California, United States
Structure (Impact Factor: 5.62). 06/2007; 15(5):535-43. DOI: 10.1016/j.str.2007.03.012
Source: PubMed


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 ( 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.

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    • "We aligned the sequences of falstatin and PbICP with those of other ICPs. Falstatin and it plasmodial homologues have long stretches of amino acids that are absent in chagasin and homologues from Leishmania mexicana and Cryptosporidium parvum [11], [12], [32] (Fig. 1). Overall, the sequence similarity between falstatin and chagasin is ∼20%, and that between falstatin and PbICP is ∼38%. "
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    ABSTRACT: Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.
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    • "However, most peptidase inhibitors are secreted and intracellular peptidase inhibitors are rare. The only examples are cystatins A and B from family I25, which inhibit cysteine peptidases 9; calpastatin, which inhibits the intracellular peptidase calpain 10; chagasin from the zooflagellate Leishmania (family I42), which also inhibits cysteine peptidases 11; three intracellular coagulation inhibitors from the horseshoe crab Tachypleus that are serpins from family I4 12; and pinA from family I24, which is an inhibitor of the ATP-dependent serine endopeptidase Lon, but is of unknown structure 13. Despite the inhibitors being intracellular, the known target peptidases are all extracellular, with the exceptions of calpain and endopeptidase Lon. "
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    ABSTRACT: We report the crystal structure solution of the Intracellular Protease Inhibitor (IPI) protein from Bacillus subtilis, which has been reported to be an inhibitor of the intracellular subtilisin Isp1 from the same organism. The structure of IPI is a variant of the all-beta, immunoglobulin (Ig) fold. It is possible that IPI is important for protein-protein interactions, of which inhibition of Isp1 is one. The intracellular nature of ISP is questioned, because an alternative ATG codon in the ipi gene would produce a protein with an N-terminal extension containing a signal peptide. It is possible that alternative initiation exists, producing either an intracellular inhibitor or a secreted form that may be associated with the cell surface. Homologues of the IPI protein from other species are multi-domain proteins, containing signal peptides and domains also associated with the bacterial cell-surface. The cysteine peptidase inhibitors chagasin and amoebiasin also have Ig-like folds, but their topology differs significantly from that of IPI, and they share no recent common ancestor. A model of IPI docked to Isp1 shows similarities to other subtilisin:inhibitor complexes, particularly where the inhibitor interacts with the peptidase active site.
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    • "The inhibitors from these families have completely different folds but still use different loops to occlude the active-site residues. In contrast to two loops in clitocypins, proteins such as cystatins, thyropins, and chagasin provide three loops, which fi ll the active-site cleft (Stubbs et al. , 1990 ; Gunč ar et al., 1999 ; Wang et al. , 2007 ; Redzynia et al. , 2008 ). "
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    ABSTRACT: Abstract Protein protease inhibitors are the tools of nature in controlling proteolytic enzymes. They come in different shapes and sizes. The β-trefoil protease inhibitors that come from plants, first discovered by Kunitz, were later complemented with representatives from higher fungi. They inhibit serine (families S1 and S8) and cysteine proteases (families C1 and C13) as well as other hydrolases. Their versatility is the result of the plasticity of the loops coming out of the stable β-trefoil scaffold. For this reason, they display several different mechanisms of inhibition involving different positions of the loops and their combinations. Natural diversity, as well as the initial successes in de novo protein engineering, makes the β-trefoil proteins a promising starting point for the generation of strong, specific, multitarget inhibitors capable of inhibiting multiple types of hydrolytic enzymes and simultaneously interacting with different protein, carbohydrate, or DNA molecules. This pool of knowledge opens up new possibilities for the exploration of their naturally occurring as well as modified properties for applications in many fields of medicine, biotechnology, and agriculture.
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