Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease.

Abstract

Recombinant human immunodeficiency virus 1 (HIV-1) protease, purified from a bacterial expression system, processed a recombinant form of its natural substrate, Pr55sag, into protein fragments that possess molecular weights commensurate with those of the virion gag proteins. Molecular weights of the protease obtained under denaturing and nondenaturing conditions (11,000 and 22,000, respectively) and chemical crosslinking studies were consistent with a dimeric structure for the active enzyme. The protease appropriately cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-ProVal-Val-NH2 between the tyrosine and proline residues. HIV-1 protease was sensitive to inactivators of the aspartic proteases. The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophenoxy)propane produced irreversible, time-dependent inactivation of the protease. The pH-dependent kinetics of this inactivator were consistent with the requirement of an unprotonated carboxyl group in the active site of the enzyme, suggesting that HIV-1 protease is also an aspartic protease.

Molecular Modeling Studies Suggest That Zinc Ions Inhibit HIV-1 Protease by Binding at Catalytic Aspartates

“…The active form of the enzyme behaves as a dimer (11,12). These findings have been confirmed by the elucidation of X-ray crystallographic structures of the enzyme both unbound (13)(14)(15) and bound to synthetic inhibitors (16)(17)(18)(19)(20).…”

“…The activity of the protease is required for viral infectivity in vitro (4). Consequently, HIV-1 PR is an attractive therapeutic target for rational drug design (5,6), and the focus of a tremendous amount of research.HIV-1 PR has been characterized as an aspartic acid protease based on sequence homology to related enzymes (7, catalytic pH studies (8), and inhibition by wellknown aspartyl protease inhibitors (9,10).The active form of the enzyme behaves as a dimer (11,12). These findings have been confirmed by the elucidation of X-ray crystallographic structures of the enzyme both unbound (13-15) and bound to synthetic inhibitors (16)(17)(18)(19)(20).…”

Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture

“…However, the striking similarities in the biological effects of this group of compounds against lentiviruses and murine C-type retroviruses may not have been so predictable. Overall, HIV and the MuLVs display limited sequence homology except for two highly conserved domains, one of which encompasses the protease enzyme's active site (12,16,20). In the proteases of these viruses, as in all known retroviral proteases, the active site includes the prototypic Asp-Thr-Gly sequence, from which the aspartic proteases derive their name (12,16,20).…”

“…Retroviral proteases, which form homodimers in the ac-tive state (20,23,25,26,35), belong to the class of aspartic proteases, all of which contain a pair of highly conserved regions composed of two hydrophobic amino acids followed by the characteristic sequence Asp-Thr/Ser-Gly (12,16,20).…”

Some highlights of virus research in 1989

“…Miller et al (1989a) reported a structure for the proteinase of Rous sarcoma virus (RSV); both Navia et al (1989) and Lapatto et al (1989) reported structures for HIV-1 proteinase; and Wlodawer et al (1989) reported a structure for a synthetic HIV-1 proteinase (in which the cysteine residues were replaced by c~-amino-n-butyrate). The material for these analyses was obtained by three methods: the RSV enzyme was extracted from virions; Navia et al and Lapatto et al utilized Various, studies, including the crystallographic analyses, indicate that the active form of the proteinase is a dimer (see also, for instance, Meek et al, 1989). As had previously been proposed, the retroviral proteinases can be seen from their folding pattern to belong to the family of aspartic proteases, such as pepsin; these are so called because their active site contains two essential aspartate residues, each in the characteristic sequence Asp-Thr(or Ser)-Gly.…”