Specificity Determinants of Human Cathepsin S Revealed by Crystal Structures of Complexes^,

Abstract

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.

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

“…Cathepsin S is homologous to the other endopeptidase cathepsins as demonstrated by the superposition of its structure onto those of cathepsins K, V, and L with root mean square deviation values of Ͻ1 Å (54). Cathepsin S showed a preference for Leu in the P 2 position in both sublibraries with a broad specificity for the P 3 position (Fig.…”

“…A characteristic of the C1 family of peptidases is its strong dependence on the S 2 subsite with the nature of the P 2 residue of the substrate being the main factor in determining the specificity of the protease (54). Most C1 family cysteine proteases prefer hydrophobic aliphatic or aromatic residues in the P 2 position (55-59).…”

“…3D), with considerably less acceptance of P 2 Pro substrates. Prior studies have attributed the preference for large aromatic and bulky residues at the P 2 site to the presence of the small residue Ala 205 (papain numbering) at the bottom of the S 2 pocket, which would suggest the presence of a large open-ended pocket (54,58). In cathepsin K, this residue is a Leu, a change that is implicated in the differential acceptance of P 2 Pro residues between the two enzymes (68).…”

Cathepsin S: therapeutic, diagnostic, and prognostic potential

“…Therefore, inhibitors require significant optimisation in order to identify a compound that displays sufficient selectivity. The determination of cathepsin S: inhibitor complex structures by X-ray crystallography, has allowed for detailed analysis of the substrate binding cleft of the protease active site (McGrath et al, 1998;Pauly et al, 2003). From this pioneering work, it is now known that cathepsin inhibitor selectivity can be developed via the optimal occupation of the S2 and S3 sub-sites, as well as overcoming gatekeeper residue interactions into the pockets (Turk et al, 2012b).…”

Contribution of Active Site Residues to Substrate Hydrolysis by USP2: Insights into Catalysis by Ubiquitin Specific Proteases

“…This mirrors to some extent the conformational duality of aldehyde inhibitor adducts with related papain-like cysteine proteases. 37 It is the TI of the acylation step in which the oxyanion must be accommodated in the oxyanion hole due to steric constraints (the bulky NH-R leaving group of the substrate does not fit into the oxyanion hole). Hence, oxyanion hole mutations can affect the overall catalysis, but their effect will likely be reduced if acylation is not the rate-limiting step.…”