Crystal Structures of Recombinant Rat Cathepsin B and a Cathepsin B-Inhibitor Complex

Abstract

The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an important role in protein catabolism and has also been implicated in various disease states. The crystal structures of two forms of native recombinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme. The structure of the native enzyme containing an underivatized active site cysteine (Cys29) showed the active enzyme conformation to be similar to that determined previously for the oxidized form. In a second structure Cys29 was derivatized with the reversible blocking reagent pyridyl disulfide. In this structure large side chain conformational changes were observed for the two key catalytic residues Cys29 and His199, demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cathepsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and inhibitor. In particular the interaction between Glu245 and the P2 Arg residue was clearly demonstrated, and it was found that the benzyl group of the P1 substrate residue occupies a large hydrophobic pocket thought to represent the S'1 subsite. This may have important implications for structure-based design of cathepsin B inhibitors.

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

“…We also observed cleavage of substrates containing a P 2 Lys residue. The efficient cleavage of P 2 ϭ Lys substrates can be attributed to the presence of Glu 205 in the P 2 pocket (papain numbering), which can mediate the charge of the basic residues via salt bridging (63)(64)(65). Cathepsin B demonstrated specificity for Lys, Leu, and Pro in the P 3 position, which is consistent with literature reports (59,66).…”

Crystal structures of human procathepsin B at 3.2 and 3.3 Å resolution reveal an interaction motif between a papain‐like cysteine protease and its propeptide

“…The crystal structures of human and rat cathepsin B [13,14] have confirmed high structural similarity to plant enzyme papain [16] and structural reasons for cathepsin B peptidyl dipeptidase activity [13,15] have also been revealed. An understanding of the molecular basis for the activation mechanism [17 19] of cathepsin B requires knowledge of the 3-dimensional structure of the proenzyme.…”

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

“…5B), the accommodation of the positive charges in S 2 could be explained by the presence of Gln 217 at the top of the pocket, which may act as a hydrogen bond donor. Such stabilization is observed in cathepsin B, which carries an Asp in this position for charge compensation (64,65). Downstream of the scissile bond, the dionain-1 S 1 Ј pocket is very similar to that of papain and is formed by the side chains of Ala 142 , Gln 148 , Asp 164 , and Trp…”

“…8A), most likely due the absence of interactions with the enzyme to restrict its motion. In papain, Tyr 61 and Tyr 67 hold the guanidinium group in place through bonding via the side-chain hydroxyl groups (61); however, dionain-1 differs by having Arg 64 and Thr 70 in the corresponding positions. This arrangement indicates clear differences with a possible effect on substrate recognition profiles from S 3 subsites and onward between these homologous cysteine proteases.…”