Capturing the Reaction Pathway in Near-Atomic-Resolution Crystal Structures of HIV-1 Protease (original) (raw)
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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. †
Proceedings of the National Academy of Sciences, 2006
HIV-1 protease is an effective target for designing drugs against AIDS, and structural information about the true transition state and the correct mechanism can provide important inputs. We present here the three-dimensional structure of a bi-product complex between HIV-1 protease and the two cleavage product peptides AETF and YVDGAA. The structure, refined against synchrotron data to 1.65 Å resolution, shows the occurrence of the cleavage reaction in the crystal, with the product peptides still held in the enzyme active site. The separation between the scissile carbon and nitrogen atoms is 2.67 Å, which is shorter than a normal van der Waal separation, but it is much longer than a peptide bond length. The substrate is thus in a stage just past the G'Z intermediate described in Northrop's mechanism [Northrop DB (2001) Acc Chem Res 34:790-797]. Because the products are generated in situ, the structure, by extrapolation, can give insight into the mechanism of the cleavage reaction. Both oxygens of the generated carboxyl group form hydrogen bonds with atoms at the catalytic center: one to the OD2 atom of a catalytic aspartate and the other to the scissile nitrogen atom. The latter hydrogen bond may have mediated protonation of scissile nitrogen, triggering peptide bond cleavage. The inner oxygen atoms of the catalytic aspartates in the complex are 2.30 Å apart, indicating a low-barrier hydrogen bond between them at this stage of the reaction, an observation not included in Northrop's proposal. This structure forms a template for designing mechanism-based inhibitors.
Journal of Molecular Biology, 2004
The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PR V82A and PR I84V that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10-1.53 Å . The crystal structures of PR and PR I84V with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PR V82A complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PR V82A showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10 Å resolution structure of PR V82A with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism.
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.
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.
Atomic resolution crystal structures of HIV-1 protease and mutants V82A and I84V with saquinavir
Proteins: Structure, Function, and Bioinformatics, 2007
Saquinavir (SQV), the first antiviral HIV-1 protease (PR) inhibitor approved for AIDS therapy, has been studied in complexes with PR and the variants PR I84V and PR V82A containing the single mutations I84V and V82A that provide resistance to all the clinical inhibitors. Atomic resolution crystal structures (0.97-1.25 Å ) of the SQV complexes were analyzed in comparison to the protease complexes with darunavir, a new drug that targets resistant HIV, in order to understand the molecular basis of drug resistance. PR I84V and PR V82A complexes were obtained in both the space groups P2 1 2 1 2 and P2 1 2 1 2 1 , which provided experimental limits for the conformational flexibility. The SQV interactions with PR were very similar in the mutant complexes, consistent with the similar inhibition constants. The mutation from bigger to smaller amino acids allows more space to accommodate the large group at P1 0 of SQV, unlike the reduced interactions observed in darunavir complexes. The residues 79-82 have adjusted to accommodate the large hydrophobic groups of SQV, suggesting that these residues are intrinsically flexible and their conformation depends more on the nature of the inhibitor than on the mutations in this region. This analysis will assist with development of more effective antiviral inhibitors. Proteins 2007;67:232-242. V V C 2007 Wiley-Liss, Inc.
X-ray structure of HIV1 protease in situ product complex
Protein Science, 2009
HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-Å resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid. Proteins 2009. © 2008 Wiley-Liss, Inc.
Protein …, 1992
Crystal structure of a complex of HIV-1 protease ... NARMADA THANKI, ' JK MOHANA RAO, ' STEPHEN I. FOUNDLING,'s3 W. JEFFREY HOWE,' JOSEPH B. MOON,' JOHN 0. HUI,2 ALFRED0 G. TOMASSELLI,' ROBERT L. HEINRIKSON,2 SUVIT THAISRIVONGS,2 AND ...