Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases

Abstract

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

Keywords: compiled kinetic data; cysteine proteases inhibitors; mechanisms of inhibition; structural-based inhibition.

Figure 1

Inhibitors of papain-like and related proteases. Complexes are shown with the same view across the active site cleft and the same scale after superimposition of proteases to cathepsin L in the p41 fragment complex. Figure was prepared using MAIN [35] and rendered with Raster3d [36]. (a) Stefin B papain complex ([21], PDB code 1STF). The stefin B chain is shown as a blue coil on the semitransparent background of the white surface of papain. (b) Inhibitor of cysteine protease (ICP) (falstatin) falcipain complex ([37], PDB code 3PNR). ICP, also known as falstatin from Plasmodium berghei, is shown as a cyan coil on the semitransparent background of the white surface of falcipain-2. (c) Chagasin cathepsin L complex ([38], PDB code 2NQD). The chagasin chain is shown as a green coil on the semitransparent background of the white surface of cathepsin L. (d) p41 fragment cathepsin L complex ([39], PDB code 1ICF). p41 fragment chain shown as a red coil on the semitransparent background of the white surface of cathepsin L. The three disulfide bonds of the p41 fragment are shown as yellow sticks. (e) Clitocypin cathepsin V complex ([40], PDB code 3H6S). The clitocypin chain is shown as a yellow coil on the semitransparent background of the white surface of cathepsin V. (f) Staphostatin staphopain complex ([41], PDB code 1PXV). The staphostatin chain is shown as a green coil on the semitransparent background of the white surface of staphopain.

Figure 2

Inhibitors of caspases. Complexes are shown with the same view and scale aligned after superimposition of the caspases to the caspase-3 in the complex with X-linked inhibitor of apoptosis (XIAP). Figure was prepared using MAIN [35] and rendered with Raster3d [36]. (a) Human XIAP caspase-3 complex ([83], PDB code 1I3O). The XIAP chain is shown as a red coil on the semitransparent background of the white surface of caspase-3. (b) Escherichia coli Nlef caspase-9 complex ([84], PDB code 3V3K). The Nlef chain is shown as an orange coil on the semitransparent background of the white surface of caspase-9. (c) Baculovirus p35 caspase-8 complex ([85], PDB code 1I4E). The p35 chain is shown as a blue coil on the semitransparent background of the white surface of caspase-8. (d) The CrmA chain ([86], PDB code 1F0C) is shown as a blue coil with the cleaved residues A359 and S359A shown as stick model in red.

Figure 3

Other inhibitors. Complexes of inhibitors are shown in views and scales adjusted to each complex and size. Figure was prepared using MAIN [35] and rendered with Raster3d [36]. (a) Procathepsin B ([98], PDB code 3PBH). The chain of cathepsin B propeptide is shown as a blue coil on the white surface of mature enzyme part of the structure. The surface part corresponding to the catalytic pair of C29 H199 residues is colored red. (b) Cystain E legumain complex ([42], PDB code 4N6N). Cystatin E is shown as a blue ribbon, with the P1 residue N39 side chain bound in to the legumain S1 site shown as a red stick model. Legumain is shown as a white surface with the S1 binding pocket colored orange and the part corresponding to the reactive site residues C189 H148 colored red. (c) Calpastatin m-calpain complex ([99], PDB code 3DF0). Calpastatin is shown as a blue surface with the loop out region indicating the position above the reactive site of calpain shown in red. Calpain-m is shown as a semitransparent white surface. (d) Securin separase complex ([100], PDB code 5ULS, 5ULT). Securin is shown as a blue surface with the region from 262 to 265 bound above the reactive site of separase shown in red. Securin is shown as a semitransparent white surface.

Figure 4

Substrate and substrate-like binding. Peptidyl substrates with their positions marked are shown as ball (nitrogen: blue, oxygens: red, carbons: cyan) and stick (cyan) models on the background of the protease surface. The surface is white with the exception of the substrate binding sites S3, S1, and S2′, respectively corresponding to substrate positions P3, P1, and P2′ colored orange, and S4, S2, and S1′ respectively corresponding to substrate positions P4, P2, and P1′ colored red. The figure was prepared using MAIN [35] and rendered with Raster3d [36]. (a) Canonical conformation of BPTI peptide bound to trypsin ([24], PDB code 2TGP). BPTI peptide is shown as a ball and stick model on the background of trypsin structure shown in white, with the exception of the substrate-binding sites surface from S3 to S3′ colored alternatively red and orange. (b) Substrate model bound to cathepsin L. The cathepsin L model was used from a previous study [11]. (c) Peptidyl inhibitor bound to legumain ([170], PDB code 4AWB). Z-Ala-Ala-Asn (ZAAN) binds to the non-prime region of the active site cleft. (d) Calpastatin loop out region bound to calpain-m ([99], PDB code 3DF0). The 172–185 region of calpastatin is shown as a coil for the main chain trace, and a ball and stick model for side chains on the background of the protease surface. The calpain surface was generated with the residues from S241 to V253, I260, and Q261 excluded to enable the view in the active site cleft. The surface of the reactive site residues C105S and H262 is purple.