Crystal structure of NS-134 in complex with bovine cathepsin B: a two-headed epoxysuccinyl inhibitor extends along the entire active-site cleft

Abstract

The crystal structure of the inhibitor NS-134 in complex with bovine cathepsin B reveals that functional groups attached to both sides of the epoxysuccinyl reactive group bind to the part of active-site cleft as predicted. The -Leu-Pro-OH side binds to the primed binding sites interacting with the His110 and His111 residues with its C-terminal carboxy group, whereas the -Leu-Gly-Meu (-Leu-Gly-Gly-OMe) part (Meu, methoxycarbonylmethyl) binds along the non-primed binding sites. Comparison with the propeptide structures of cathepsins revealed that the binding of the latter part is least similar to the procathepsin B structure; this result, together with the two-residue shift in positioning of the Leu-Gly-Gly part, suggests that the propeptide structures of the cognate enzymes may not be the best starting point for the design of reverse binding inhibitors.

Figure 1. Schematic representation of the epoxysuccinyl inhibitors E-64, CA030, CA074, NS-134 and CLIK148

Figure 2. Structure of the complex of cathepsin B with NS-134

The cathepsin B molecule is presented in cyan (sticks) and the inhibitor NS-134 in yellow. Oxygen and nitrogen atoms are shown in red and blue respectively. Hydrogen bonds are shown in white dotted lines. Amino acid residues of the cathepsin B molecule around the active-site cleft that interact with the substrate are represented by a ball-and-stick model in green. The Figure was prepared with MAIN [34] and rendered with Raster 3D [46]. The molecular surface was generated with GRASP [47].

Figure 3. Active-site cleft of cathepsin B with NS-134 and the superimposed inhibitors CA030 and E-64

The surface of cathepsin B molecule is presented in white. NS-134 is presented in dark grey, CA030 (1CSB; [14]) is shown in grey and E-64 (1ATK; [39]) in light grey. The Figure was prepared with MAIN [34] and rendered with Raster 3D [46]. The molecular surface was generated with GRASP [47].

Figure 4. Structure of the cathepsin B–NS-134 complex and a closer view of the active-site cleft, showing the superimposed propeptide part (3PBH; [22])

NS-134 is represented as a ball-and-stick model in dark grey. The propeptide part is shown in light grey. The Figure was prepared with MAIN [34] and rendered with Raster 3D [46]. The molecular surface was generated with GRASP [47].

Figure 5. Cathepsin B molecule (white surface) from the cathepsin B–NS-134 complex (the inhibitor is represented as a ball-and-stick model in dark grey), with the superimposed propeptide parts from procathepsins X (1DEU; [42]), B (3PBH; [22]), L (1CJL; [40]) and K (1BY8; [41]) shown in dark grey, light grey and white respectively

The Figure was prepared with MAIN [34] and rendered with Raster 3D [46]. The molecular surface was generated with GRASP [47].