No abstract

Cathepsin L is a member of the papain superfamily of cysteine proteases and, like many other proteases, it is synthesized as an inactive proenzyme. Its prosegment shows little homology to that of procathepsin B, whose structure, the first for a cysteine protease proenzyme, has been determined recently. We report here the 3‐D structure of a mutant of human procathepsin L determined at 2.2 A resolution, describe the mode of binding employed by the prosegment and discuss the molecular basis for other possible roles of the prosegment. The N‐terminal part of the prosegment is globular and contains three alpha‐helices with a small hydrophobic core built around aromatic side chains. This domain packs against a loop on the enzyme's surface, with the aromatic side chain from the prosegment being located in the center of this loop and providing a large contact area. The C‐terminal portion of the prosegment assumes an extended conformation and follows along the substrate binding cleft toward the N‐terminus of the mature enzyme. The direction of the prosegment in the substrate binding cleft is opposite to that of substrates. The previously described role of the prosegment in the interactions with membranes is supported by the structure of its N‐terminal domain. The fold of the prosegment and the mechanism by which it inhibits the enzymatic activity of procathepsin L is similar to that observed in procathepsin B despite differences in length and sequence, suggesting that this mode of inhibition is common to all enzymes from the papain superfamily.

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole d o not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap. Keywords: crystallography; interfacial activation; lipasesLipases of known 3-dimensional structure show significant similarities in their topologies and conform in full or in part to the a/fl hydrolase fold ( Ollis et al., 1992). The catalytic machinery of lipases consists of a serine protease-like triad, Ser-His-Asp/Glu, and their hydrolysis of ester bonds of triacylglycerols is thought to involve an enzymatic mechanism similar to that of the serine proteases (Chapus et al., 1988). The activity of lipases is dramatically enhanced by the presence of a lipid/water interface (Desnuelle, 1972), and it is now well accepted that the phenomenon of interfacial activation involves a conformational change in the enzyme. This has been documented for 2 lipases, one from Rhizomucor miehei (Brzozowski et ai., 199i) and another from human pancreas (van Tilbeurgh et al., 1993), in which binding of an inhibitor or substrate analog causes a conformational rearrangement of 1 or more loops near the active site, exposing the serine nucleophile to the solvent and creating also an oxyanion hole in the process.A similar rearrangement was also proposed for larger lipases from Geotrichum candidurn (GCL;Schrag & Cygler, 1993) Candida rugosa (CRL; Grochubki et ai., 1993). These 2 enzymes show -40% amino acid sequence identity, and their overall 3D structures are very similar (Grochulski ...

The crystal structure of a Ca(2+)-binding domain (dVI) of rat m-calpain has been determined at 2.3 A resolution, both with and without bound Ca2+. The structures reveal a unique fold incorporating five EF-hand motifs per monomer, three of which bind calcium at physiological calcium concentrations, with one showing a novel EF-hand coordination pattern. This investigation gives us a first view of the calcium-induced conformational changes, and consequently an insight into the mechanism of calcium induced activation in calpain. The crystal structures reveal a dVI homodimer which provides a preliminary model for the subunit dimerization in calpain.