Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

Abstract

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives

“…These results are consistent with our previous observations when screening chalcones for cathepsin L inhibition where we found that Fh CL1 activity was easier to disturb and was inhibited by a higher number of the compounds assayed when compared to Fh CL3 [16]. This could be a consequence of the broader conformation of Fh CL1 active site cleft that might accommodate different small molecules straightforwardly [33,34].…”

Low allelic diversity in vaccine candidates genes from different locations sustain hope for Fasciola hepatica immunization

“…Val is present in FhCL1 at this position, the presence of Leu is characteristic of FhCL2. However, a recent study demonstrates that a single change in this position generated by site directed mutagenesis does not affect the specificity of the enzyme (Corvo et al, 2018). These observations reinforce the idea that a diversity of FhCL1 isoforms with subtle differences might be produced simultaneously by the parasite.…”

Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development

“…The main viral protease, 3CLpro, is a cysteine protease with LQ/(S, A, G) as the preferred P2–P1–P1’ substrate sequence ( Fan, Ma et al 2005 ). In comparison, the optimum substrate recognition motif for rhinovirus 3C protease is ETLGQ/GP ( Cordingley, Callahan et al 1990 ), highlighting the broad range in substrate recognition among cysteine proteases ( Corvo, Ferraro et al 2018 ). Due to its key role in viral replication, 3CLpro is considered a major target for antiviral drug discovery.…”