Substrate Specificity of Cysteine Proteases Beyond the S Pocket: Mutagenesis and Molecular Dynamics Investigation of Cathepsins L.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous 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 S subsite (pocket) of the enzyme that interacts with the substrate P residue (Schetcher and Berger nomenclature). Although secreted CL1 accommodates aliphatic residues in the S pocket, CL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the CL1 S subsite at three amino acid positions to render it identical to that present in CL2. The substitutions did not produce the expected increment in proline accommodation in P Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S and S subsites that contributes to the accommodation of Gly in S. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S and S pockets of the mutant CL1 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.