Atomic resolution crystal structures of HIV-1 protease and mutants V82A and I84V with saquinavir (original) (raw)
Related papers
Journal of Molecular Biology, 2005
The crystal structures, dimer stabilities, and kinetics have been analyzed for wild-type human immunodeficiency virus type 1 (HIV-1) protease (PR) and resistant mutants PR L24I , PR I50V , and PR G73S to gain insight into the molecular basis of drug resistance. The mutations lie in different structural regions. Mutation I50Valters a residue in the flexible flap that interacts with the inhibitor, L24I alters a residue adjacent to the catalytic Asp25, and G73S lies at the protein surface far from the inhibitor-binding site. PR L24I and PR I50V , showed a 4% and 18% lower k cat /K m , respectively, relative to PR. The relative k cat /K m of PR G73S varied from 14% to 400% when assayed using different substrates. Inhibition constants (K i ) of the antiviral drug indinavir for the reaction catalyzed by the mutant enzymes were about threefold and 50-fold higher for PR L24I and PR I50V , respectively, relative to PR and PR G73S . The dimer dissociation constant (K d ) was estimated to be approximately 20 nM for both PR L24I and PR I50V , and below 5 nM for PR G73S and PR. Crystal structures of the mutants PR L24I , PR I50V and PR G73S were determined in complexes with indinavir, or the p2/NC substrate analog at resolutions of 1.10-1.50 Å. Each mutant revealed distinct structural changes relative to PR. The mutated residues in PR L24I and PR I50V had reduced intersubunit contacts, consistent with the increased K d for dimer dissociation. Relative to PR, PR I50V had fewer interactions of Val50 with inhibitors, in agreement with the dramatically increased K i . The distal mutation G73S introduced new hydrogen bond interactions that can transmit changes to the substrate-binding site and alter catalytic activity. Therefore, the structural alterations observed for drug-resistant mutations were in agreement with kinetic and stability changes.
Proteins: Structure, Function, and Genetics, 2001
Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both complex and varied. We have determined crystal structures of HIV-1 protease mutants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogues of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to study the structural mechanisms underlying resistance. The structures were determined at 1.55-1.9-Å resolution and compared with the wild-type structure. The conformational disorder seen for most of the hydrophobic side-chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location suggests that they are important for catalysis as well as structural stability. Structural differences among the mutants were analyzed in relation to the observed changes in protease activity and stability. Mutant L90M shows steric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stability and activity. Mutant K45I reduces the mobility of the flap and the inhibitor and contributes to an enhancement in structural stability and activity. The side-chain variations at residue 30 relative to wildtype are the largest in D30N and the changes are consistent with the altered activity observed with peptide substrates. Polar interactions in D30N are maintained, in agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D. Proteins 2001;43:455-464.
FEBS Journal, 2005
HIV-1 protease (PR) and two drug-resistant variants -PR with the V82A mutation (PR V82A ) and PR with the I84V mutation (PR I84V ) -were studied using reduced peptide analogs of five natural cleavage sites (CA-p2, p2-NC, p6 pol -PR, p1-p6 and NC-p1) to understand the structural and kinetic changes. The common drug-resistant mutations V82A and I84V alter residues forming the substratebinding site. Eight crystal structures were refined at resolutions of 1.10-1.60 Å. Differences in the PR-analog interactions depended on the peptide sequence and were consistent with the relative inhibition. Analog p6 pol -PR formed more hydrogen bonds of P2 Asn with PR and fewer van der Waals contacts at P1′ Pro compared with those formed by CA-p2 or p2-NC in PR complexes. The P3 Gly in p1-p6 provided fewer van der Waals contacts and hydrogen bonds at P2-P3 and more water-mediated interactions. PR I84V showed reduced van der Waals interactions with inhibitor compared with PR, which was consistent with kinetic data. The structures suggest that the binding affinity for mutants is modulated by the conformational flexibility of the substrate analogs. The complexes of PR V82A showed smaller shifts of the main chain atoms of Ala82 relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. PR V82A was able to compensate for the loss of interaction with inhibitor caused by mutation, in agreement with kinetic data, but substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation. Hence, these structures help to explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.
European Journal of Biochemistry, 2004
The crystal structures of the wild-type HIV-1 protease (PR) and the two resistant variants, PR V82A and PR L90M , have been determined in complex with the antiviral drug, indinavir, to gain insight into the molecular basis of drug resistance. V82A and L90M correspond to an active site mutation and nonactive site mutation, respectively. The inhibition (K i ) of PR V82A and PR L90M was 3.3-and 0.16fold, respectively, relative to the value for PR. They showed only a modest decrease, of 10-15%, in their k cat /K m values relative to PR. The crystal structures were refined to resolutions of 1.25-1.4 Å to reveal critical features associated with inhibitor resistance. PR V82A showed local changes in residues 81-82 at the site of the mutation, while PR L90M showed local changes near Met90 and an additional interaction with indinavir. These structural differences concur with the kinetic data.
Biochemical and Biophysical Research Communications, 2010
The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.
Proceedings of the National Academy of Sciences, 1993
The crystal structure of HIV-2 protease in complex with a reduced amide inhibitor [BI-LA-398; Phe-Val-Phe-psi (CH2NH)-Leu-Glu-Ile-amide] has been determined at 2.2-A resolution and refined to a crystallographic R factor of 17.6%. The rms deviation from ideality in bond lengths is 0.018 A and in bond angles is 2.8 degrees. The largest structural differences between HIV-1 and HIV-2 proteases are located at residues 15-20, 34-40, and 65-73, away from the flap region and the substrate binding sites. The rms distance between equivalent C alpha atoms of HIV-1 and HIV-2 protease structures excluding these residues is 0.5 A. The shapes of the S1 and S2 pockets in the presence of this inhibitor are essentially unperturbed by the amino acid differences between HIV-1 and HIV-2 proteases. The interaction of the inhibitor with HIV-2 protease is similar to that observed in HIV-1 protease structures. The unprotected N terminus of the inhibitor interacts with the side chains of Asp-29 and Asp-30. The glutamate side chain of the inhibitor forms hydrogen bonds with the main-chain amido groups of residues 129 and 130.
Journal of Medicinal Chemistry, 2008
In our quest for HIV-1 protease inhibitors that are not affected by the V82A resistance mutation, we have synthesized and tested a second generation set of C 2 -symmetric HIV-1 protease inhibitors that contain a cyclohexane group at P1 and/or P1′. The binding affinity results indicate that these compounds have an improved response to the appearance of the V82A mutation than the parent compound. The X-ray structure of one of these compounds with the V82A HIV-1 PR variant provides the structural rationale for the better resistance profile of these compounds. Moreover, scrutiny of the X-ray structure suggests that the ring of the Cha side chain might be in a boat rather than in the chair conformation, a result supported by molecular dynamics simulations.
Biochemistry, 2007
HIV-1 protease (PR) is the target for several important antiviral drugs used in AIDS therapy. The drugs bind inside the active site cavity of PR where normally the viral polyprotein substrate is bound and hydrolyzed. We report two high-resolution crystal structures of wild-type PR (PR WT ) and the multidrug-resistant variant with the I54V mutation (PR I54V ) in complex with a peptide at 1.46 and 1.50 Å resolution, respectively. The peptide forms a gem-diol tetrahedral reaction intermediate (TI) in the crystal structures. Distinctive interactions are observed for the TI binding in the active site cavity of PR WT and PR I54V . The mutant PR I54V /TI complex has lost water-mediated hydrogen bond interactions with the amides of Ile50 and Ile50′ in the flap. Hence, the structures provide insight into the mechanism of drug resistance arising from this mutation. The structures also illustrate an intermediate state in the hydrolysis reaction. One of the gem-diol hydroxide groups in the PR WT complex forms a very short (2.3 Å) hydrogen bond with the outer carboxylate oxygen of Asp25. Quantum chemical calculations based on this TI structure are consistent with protonation of the inner carboxylate oxygen of Asp25′, in contrast to several theoretical studies. These TI complexes and quantum calculations are discussed in relation to the chemical mechanism of the peptide bond hydrolysis catalyzed by PR. †
Journal of Virology, 2003
Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.