The residues of the inhibitor are labeled without a chain designator. The residues of the enzyme are marked "e", except for the first residue, marked "p" (E1p), which belongs to the prosegment sequence.Abbreviations used: E-64, L-trans-epoxysuccinylleucylamido-(4-guanidino)butane; MAD, multiwavelength anomalous diffraction; MS, mass spectrometry; PDB, Protein Data Bank; NHMec, 7-(4-methyl)coumarylamide; r.m.s., root mean square; Z-, carboxybenzoyl-; GST,
glutathione-S-transferaseChagasin-cathepsin L complex structure 3
SUMMARY
Cathepsin B is a papain-like cysteine protease showing both endo-and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (K i for H110A cathepsin B, 0.35 nM) due to an improved association rate (k on , 5 ؋ 10 5 M ؊1 s ؊1 ). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 Å resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.
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.
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