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

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

doi:10.1016/s0021-9258(19)78106-x

Cited by 34 publications

(14 citation statements)

References 22 publications

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“…The RSV I44V Mutant CleaVes the NC-PR Peptide at Two Sites. RSV PR residue 44 (Val32 in HIV-1 PR) was previously shown to influence the S2 and S2′ subsites (5). The RSV PR I44V mutant tolerated P2 or P2′ Leu substitutions in the NC-PR peptide with at least 60% and 30% activity, respectively, relative to the unsubstituted peptide (5,7,25).…”

Section: Drug-resistantmentioning

confidence: 99%

“…and RSV PRs. Many of the amino acids that contribute to differences in substrate selection by the HIV-1 and RSV PRs were identified previously by structural and biochemical comparisons of the two enzymes (2)(3)(4)(5). (See Table 1 for a list of amino acids predicted by analysis of PR structure to influence substrate selection.)…”

Section: Rationale For Introduction Of Specific Mutations Into Hiv-1mentioning

confidence: 99%

“…The effects of PR mutations were analyzed using two peptide libraries containing substitutions of single residues from P4 to P4′. One library was based on the RSV NC-PR Gag junction sequence (PPAVS-LAM-TMRR) previously shown to distinguish changes in specificity in individual enzyme subsites (2)(3)(4)(5). The other library was based on the HIV-1 CA-NCa cleavage junction (PARVL-AEAMR).…”

Section: Rationale For Introduction Of Specific Mutations Into Hiv-1mentioning

confidence: 99%

“…RSV PR Gln63 has no HIV-1 PR structural equivalent. Sitedirected mutagenesis was used to substitute the amino acids found in HIV-1 into their structurally equivalent positions of the RSV PR (2)(3)(4)(5). The resultant RSV protease mutants, containing substitutions at one or more of these key-positions, displayed significant HIV-1-like substrate selection (2)(3)(4)(5).…”

mentioning

confidence: 99%

“…Sitedirected mutagenesis was used to substitute the amino acids found in HIV-1 into their structurally equivalent positions of the RSV PR (2)(3)(4)(5). The resultant RSV protease mutants, containing substitutions at one or more of these key-positions, displayed significant HIV-1-like substrate selection (2)(3)(4)(5). Additionally, a mutant known as RSV S9 PR, which combined all of the above substitutions except at residues Gln63 and His65, and whose structure has been solved (6), became specifically susceptible to inhibition by HIV-1 protease inhibitors at concentrations too low to inhibit wildtype RSV protease (7).…”

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confidence: 99%

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Drug-Resistant HIV-1 Proteases Identify Enzyme Residues Important for Substrate Selection and Catalytic Rate

et al. 1998

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A series of mutations, first identified in protease inhibitor-resistant HIV-1 viral isolates, were introduced into HIV-1 PR as individual substitutions. Mutants containing R8K, V32I, V82T, I84V, G48V/L90M, or V82T/I84V substitutions were analyzed for differences in substrate preference and catalytic efficiency using a set of single amino acid substituted HIV-1 CA-NCa cleavage site peptides. All mutants exhibited wild-type preference for large hydrophobic residues, especially Phe, in the P1' substrate position. Only the R8K and V32I mutants showed significant differences in subsite selection compared to wild-type enzyme. In a parallel study, the individual mutations R10K, L12V, I44V, A60M, I71V, and I108V were introduced into RSV PR. These amino acid positions are structurally equivalent to Arg8, Leu10, Val32, Met46, Ile54, and Ile84 in HIV-1 PR, respectively, which mutate in drug-resistance. The RSV R10K substitution significantly altered substrate specificity and catalytic rate, compared to wild-type, in a manner similar to that of the HIV-1 R8K mutant. Crystal structures of the RSV PR R10K, I44V, I71V, and Il08V mutant enzymes presented here indicate that each of these substitutions has little effect on the overall structure of the respective enzymes. Taken together, these data provide an explanation for the reported in vivo predilection for selection of large hydrophobic residues in the P1' substrate position of second locus mutations in the Gag polyprotein PR cleavage sites. The data also suggest that the selection of resistant enzymes is not simply limited to loss of binding to inhibitor but affects other steps in proteolysis.

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Section: Drug-resistantmentioning

confidence: 99%

Section: Rationale For Introduction Of Specific Mutations Into Hiv-1mentioning

confidence: 99%

Section: Rationale For Introduction Of Specific Mutations Into Hiv-1mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Drug-Resistant HIV-1 Proteases Identify Enzyme Residues Important for Substrate Selection and Catalytic Rate

et al. 1998

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Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

et al. 2012

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HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

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Proteolytic Processing and Particle Maturation

Vogt

1996

Current Topics in Microbiology and Immunology

126

110

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

Cited by 34 publications

References 22 publications

Drug-Resistant HIV-1 Proteases Identify Enzyme Residues Important for Substrate Selection and Catalytic Rate

Drug-Resistant HIV-1 Proteases Identify Enzyme Residues Important for Substrate Selection and Catalytic Rate

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

Proteolytic Processing and Particle Maturation

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