Crystal structure of the parasite inhibitor chagasin in complex with papain allows identification of structural requirements for broad reactivity and specificity determinants for target proteases

Abstract

Papain (EC 3.4.22.2) from the latex of the papaya fruit (Carica papaya) was one of the first known proteolytic enzymes, and its digestive properties were already being utilized in the 19th century. Detailed biochemical studies in the 20th century peaked with efforts in the 1960s, defining the chemistry of the enzymatic mechanism, delineating the concept of specificity for protein substrate recognition [1][2][3], and with elucidation of the A complex of chagasin, a protein inhibitor from Trypanosoma cruzi, and papain, a classic family C1 cysteine protease, has been crystallized. Kinetic studies revealed that inactivation of papain by chagasin is very fast (k on = 1.5 · 10 6 m )1 AEs )1 ), and results in the formation of a very tight, reversible complex (K i = 36 pm), with similar or better rate and equilibrium constants than those for cathepsins L and B. The high-resolution crystal structure shows an inhibitory wedge comprising three loops, which forms a number of contacts responsible for the high-affinity binding. Comparison with the structure of papain in complex with human cystatin B reveals that, despite entirely different folding, the two inhibitors utilize very similar atomic interactions, leading to essentially identical affinities for the enzyme. Comparisons of the chagasin-papain complex with high-resolution structures of chagasin in complexes with cathepsin L, cathepsin B and falcipain allowed the creation of a consensus map of the structural features that are important for efficient inhibition of papain-like enzymes. The comparisons also revealed a number of unique interactions that can be used to design enzyme-specific inhibitors. As papain exhibits high structural similarity to the catalytic domain of the T. cruzi enzyme cruzipain, the present chagasin-papain complex provides a reliable model of chagasin-cruzipain interactions. Such information, coupled with our identification of specificity-conferring interactions, should be important for the development of drugs for treatment of the devastating Chagas disease caused by this parasite.

Solution Structure of a Phytocystatin from Ananas comosus and Its Molecular Interaction with Papain

“…Three structural elements essentially responsible for the specific binding of cystatins to the active-site cleft of papain-like cysteine proteases are a conserved Gly at the N-terminus, a QxVxG motif situated in the β-hairpin loop between the second and the third strand, and an aromatic residue on the β-hairpin loop between the fourth and the fifth strand [13], [14], [15]. The parasite chagasins, cystatin-like proteins were newly found to have a similar recognition pattern with cysteine proteases [16], [17], [18], [19], [20]. Although chagasins strongly inhibit cysteine proteases, the three inhibitory loops of chagasins show low sequence homology to other cystatins.…”

Host cell death induced by the egress of intracellular Plasmodium parasites

“…Interestingly, it has previously been demonstrated that Plasmodium parasites express a very potent chagasinlike cysteine protease inhibitor called falstatin, which has the capacity to block caspase activity [25]. This is very surprising since chagasin-like inhibitors are known to normally bind to conserved grooves of papain-like proteases [26,27] and caspases do not have such a groove [28].…”

Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

“…In our docking model, the phenyl ring of EhICP2 Phe80 protrudes into the active site of papain. Redzynia and coworkers postulated that residue Thr31 of the chagasin BC loop is an "active-site-blocking residue", which interacts with the active site of papain (Redzynia et al, 2009), and also binds the catalytic cysteine of falcipain 2 by a water-assisted hydrogen bond (Wang et al, 2007). The corresponding residue in EhICP2 is Ser47.…”

“…To gain insights into the binding mechanisms of EhICP2 to a target protease, the structure of EhICP2 was superimposed onto the coordinates of chagasin from the papain-chagasin complex (Redzynia et al, 2009). To dock EhICP2 onto papain, we used the fiberdock program, which minimizes the EhICP2 molecule and favors ligand backbone flexibility as the determinant parameter in docking (Mashiach et al, 2010).…”

“…Crystal and NMR structures of ICPs from T. cruzi and L. mexicana demonstrated that ICPs adopt an immunoglobulin-like fold composed of seven β strands, one 3 10 -helix, and three loops named BC, DE, and FG (Smith et al, 2006;Figueiredo da Silva et al, 2007). Crystal structures of chagasin in complex with falcipain (Wang et al, 2007), papain (Redzynia et al, 2009), cathepsin L (Ljunggren et al, 2007), and cathepsin B (Redzynia et al, 2008) demonstrate that inhibition by ICPs is accomplished by a precise interaction between loops BC, DE, and FG and the target cysteine protease.…”