Synthetic “interface” peptides alter dimeric assembly of the HIV 1 and 2 proteases

Abstract

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N-and C-terminal residues of both monomers, which form a four-stranded 6-sheet. Peptides mimicking one 6-strand (residues 95-99), or two &strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIVl) interface were shown to inhibit the HIVl and 2 proteases (PRs) with ICso's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIVl P R dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIVl PR. Both one-and two-strand peptides alter the monomer/dimer equilibrium of both HIVl and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIVl and H1V2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one-and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.Keywords: aspartyl protease; cross-linking; dimer interface; dissociation; enzyme inhibition; HIVl protease; HIV2 protease; peptide; refolding; tethered dimer Dimerization is required to create a symmetric active site for aspartyl proteases (PRs) encoded by retroviruses. The crystal structures of the human immunodeficiency virus 1 (HIVl) (Navia et al., 1989;Wlodawer et al., 1989) and Rous sarcoma virus (RSV) PRs confirmed the homodimeric nature of these viral enzymes and also defined the regions of interaction between the monomers. The extensive hydrophobic interface present in these PR dimers is dominated by a n antiparallel P-sheet formed by the interdigitation of the N-terminal (residues 1-4 in HIVl and 1-5 in RSV) and the C-terminal (residues 96-99 in HIVl and 119-124 in RSV) 0-strands of each monomer ( Fig. 1 and kinemages). These interactions appear to be a major stabilizing force in the enzyme,

Characterization of a folding intermediate from HIV‐1 ribonuclease H

“…In dihydrofolate reductase (Hall & Frieden, 1989), isoleucyl-t-RNA synthetase (Michaels et al, 1996) and the dimeric retroviral protease (Babe et al, 1992), peptide fragments of the respective proteins specifically inhibited their correct folding and assembly. Because the C-terminal region of RNase H appears to fold late and proper folding of this region is critical for RNase H function, the intermediate we have characterized here represents a potentia1 target for such a folding inhibitor.…”

Sequence‐Specific Resonance Assignments of the 1H‐NMR Spectra and Structural Characterization in Solution of the HIV‐1 Transframe Protein p6*

“…There is evidence from in vitro transcription-translation assays that the frame shift protein p6* inhibits the processing activity of the HIV protease by blocking the active site in a manner similar to the pepsinogen N-terminal prosegment (James and Sielecki, 1986;Partin et al, 1991 ;Zybarth and Carter, 1995). On the other hand, the C-terminal hexapeptide of p6* was reported to inhibit the protease by preventing its dimerization with even larger affinity than the respective C-terminal peptide of the protease itself, which to-gether with the N-termini forms a four-stranded antiparallel psheet (BabC et al, 1992;Schramm et al, 1993;Weber, 1990).…”

Human immunodeficiency virus‐1 reverse transcriptase heterodimer stability

“…Because dimerization is required to generate a functional enzyme, it has been suggested that agents that can bind at the dimer interface and prevent subunit association could represent a novel therapeutic approach tb stem the progression of AIDS (Restle et al, 1990;Babe et al, 1992;Nanni et al, 1993). Very recently, several peptides derived from the connection domain have been shown to inhibit the dimerization of RT (Divita et al, 1994).…”