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

Craig E. Cameron, Todd W. Ridky, Sergey Shulenin, Jonathan Leis, Irene T. Weber, Terry Copeland, Alexander Wlodawer, Haim Burstein, Diane Bizub-Bender, Anna Marie Skalka

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)11170-11177
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number15
StatePublished - Apr 15 1994

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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