Displacement of the Occluding Loop by the Parasite Protein, Chagasin, Results in Efficient Inhibition of Human Cathepsin B

John S Mort,Anna Ljunggren,Grzegorz Bujacz,Joanne C Krupa,Mariusz Jaskolski,Izabela Redzynia,Magnus Abrahamson

doi:10.1074/jbc.m802064200

Abstract

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 (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 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 angstroms 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.

Highlights

In cathepsin B, a histidine residue (His-111e)4 provides a positive charge to accept the C-terminal carboxylate of a substrate
Some natural protein inhibitors of papain-like enzymes, such as some cystatins and chagasin, manage to control cathepsin B activity by relatively tight binding in the active site cleft of the enzyme
The analysis of the function of chagasin as a cathepsin B inhibitor and of its structure in cathepsin B complex reported in the present study clarifies how the inhibitor overcomes the restricted access to the active site cleft posed by the occluding loop

Summary

EXPERIMENTAL PROCEDURES

Proteins—The recombinant chagasin construct included an extra N-terminal GPLGS sequence as a cloning artifact, with the remaining sequence corresponding to the native protein (GenBankTM/EMBL accession number AJ299433) [20]. Active papain was prepared from the commercial papaya latex enzyme (Sigma-Aldrich) through affinity purification on Sepharose 4B-coupled Gly-Gly-Tyr-Arg column, as detailed previously [24, 25]. Purified this way, the enzyme could be activated to at least 65% even after storage at Ϫ80 °C for up to 3 months. Corrections for substrate competition were made using a Km value of 55 ␮M for cathepsin B hydrolysis of Z-Phe-Arg-NHMec. To calculate the association rate constants for chagasin interaction with S115A- and (H110A,S115A)-cathepsin B, the pseudofirst-order rate constants (kobs) in continuous-rate assays with different concentrations of chagasin were determined from the pre-steady-state phase of the velocity curve by non-linear regression using FLUSYS (obtained from Dr Neil Rawlings, The Babraham Institute).

PDB code

RESULTS AND DISCUSSION

Form II