Programming the Rous sarcoma virus protease to cleave new substrate sequences

The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave new substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally

Mutational analysis of the substrate binding pockets of the Rous sarcoma virus and human immunodeficiency virus-1 proteases

Mutations, designed by analysis of the crystal structures of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) protease (PR), were introduced into the substrate binding pocket of RSV PR. The mutations substituted nonconserved residues of RSV PR, located within 10 Å of the substrate, for those in structurally equivalent positions of HIV-1 PR. Changes in the activity of purified mutants were detected in vitro by following cleavage of synthetic peptides representing wild-type and modified RSV and HIV-1 gag and pol polyprotein cleavage sites. Substituting threonine for valine 104 (V104T), S107N, I44V, Q63M or deletion of residues 61-63 produced enzymes that were 2.5-7-fold more active than the wild type RSV PR. Substituting I42D, M73V, and A100L produced enzymes with lower activity, whereas a mutant that included both M73V and A100L was as active as wild type. Several substitutions altered the specificity for substrate. These include I42D and I44V, which contribute to the S2 and S2' subsites. These proteins exhibited HIV-1 PR specificity for P2- or P2'-modified peptide substrates but unchanged specificity with P4-, P3-, P1-, P1'-, and P3'- modified substrates. Changes in specificity in the S4 subsite were detected by deletion of residues 61-63. These results confirm the hypothesis that the subsites of the substrate binding pocket of the retroviral protease are capable of acting independently in the selection of substrate amino acids