Mutations that alter the activity of the rous sarcoma virus protease

Bjorn Grinde, Craig E. Cameron, Jonathan Leis*, Irene T. Weber, Alexander Wlodawer, Haim Burstein, Diane Bizub, Anna Marie Skalka

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Scopus citations


Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.

Original languageEnglish (US)
Pages (from-to)9481-9490
Number of pages10
JournalJournal of Biological Chemistry
Issue number14
StatePublished - May 15 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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