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.

Understanding the chemical physical properties of protein binding sites is at the basis of the rational design of protein ligands. The hinge region of the Fc fragment of immunoglobulin G is an important and well characterized protein binding site, known to interact with several natural proteins and synthetic ligands. Here, we report structural evidence that a Staphylococcus aureus Protein A mimetic peptide dendrimer, deduced by a combinatorial approach, binds close to the Cgamma2/Cgamma3 interface of the constant fragment of a human IgG1 molecule, partially hindering the Protein A binding site. The X-ray analysis evidenced a primary binding site located between a terminal Arg residue of the ligand peptidic arm and a hydrophobic protein site consisting of Val308, Leu309, and His310. A molecular dynamic analysis of the model derived from the X-ray structure showed that in water at room temperature the complex is further stabilized by the formation of at least one more contact between a terminal Arg residue of the second arm of the peptide and the carboxylic group of a protein amino acid, such as Glu318, Asp312, or Asp280. It appears thus that stability of the Fc-dendrimer complex is determined by the synergetic formation of multiple bonds of different nature between the dendrimer arms and the protein accessible sites. The electrostatic and van der Waals energies of the complex were monitored during the MD simulations and confirmed the energetic stability of the two interactions.