X-ray structure of HIV1 protease in situ product complex (original) (raw)
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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.
Capturing the Reaction Pathway in Near-Atomic-Resolution Crystal Structures of HIV-1 Protease
Biochemistry, 2012
Snapshots of three consecutive steps in the proteolytic reaction of HIV-1 protease (PR) were obtained in crystal structures at resolutions of 1.2−1.4 Å. Structures of wild-type protease and two mutants (PR V32I and PR I47V ) with V32I and I47V substitutions, which are common in drug resistance, reveal the gem-diol tetrahedral intermediate, the separating N-and C-terminal products, and the C-terminal product of an autoproteolytic peptide. These structures represent three stages in the reaction pathway and shed light on the reaction mechanism. The near-atomic-resolution geometric details include a short hydrogen bond between the intermediate and the outer carboxylate oxygen of one catalytic Asp25 that is conserved in all three structures. The two products in the complex with mutant PR I47V have a 2.2 Å separation of the amide and carboxyl carbon of the adjacent ends, suggesting partial cleavage prior to product release. The complex of mutant PR V32I with a single C-terminal product shows density for water molecules in the other half of the binding site, including a partial occupancy water molecule interacting with the product carboxylate end and the carbonyl oxygen of one conformation of Gly27, which suggests a potential role of Gly27 in recycling from the product complex to the ligand-free enzyme. These structural details at near-atomic resolution enhance our understanding of the reaction pathway and will assist in the design of mechanism-based inhibitors as antiviral agents.
Journal of The American Chemical Society, 2010
Structural snapshots of each step in the catalytic cycle would help development of inhibitors of human immunodeficiency virus type 1 protease (HIV-1 PR) as effective drugs against HIV/AIDS. We report here one snapshot obtained by determining the structure of enzyme-substrate complex under conditions where the catalytic activity of the enzyme is greatly reduced. The 1.76 Å crystal structure shows the oligopeptide substrate, AETFYVDGAA, converted in situ into a gem-diol tetrahedral intermediate (TI). The gem-diol intermediate is neutral and one of the hydroxyl oxygens forms a very short hydrogen bond (2.2 Å) with the anionic aspartate of the catalytic dyad, which is monoprotonated. Further, there is no hydrogen atom on the outer oxygen of the neutral aspartate. These two observations provide direct evidence that, in the reaction mechanism, hydrogen bonding between catalytic aspartate and scissile carbonyl oxygen facilitates water attack on the scissile carbon atom. Comparison with the structural snapshot of the biproduct complex involving the same substrate reveals the reorganization of the hydrogen bonds at the catalytic center as the enzymatic reaction progresses toward completion. Accumulation of TI in the crystals provides direct evidence that collapse of TI is the rate-limiting step of hydrolysis.
Three-Dimensional Structures of HIV-1 and SIV Protease Product Complexes † , ‡
Biochemistry, 1996
Strain is eliminated as a factor in hydrolysis of the scissile peptide bond by human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), based on the first eight complexes of products of hydrolysis with the enzymes. The carboxyl group generated at the scissile bond interacts with both catalytic aspartic acids. The structures directly suggest the interactions of the gemdiol intermediate with the active site. Based on the structures, the nucleophilic water is displaced stereospecifically by substrate binding toward one catalytic aspartic acid, while the scissile carbonyl becomes hydrogen bonded to the other catalytic aspartic acid in position for hydrolysis. Crystal structures for two N-terminal (P) products and two C-terminal (Q) products provide unambiguous density for the ligands at 2.2-2.6 Å resolution and 17-21% R factors. The N-terminal product, Ac-S-L-N-F/, overlaps closely with the N-terminal sequences of peptidomimetic inhibitors bound to the protease. Comparison of the two C-terminal products, /F-L-E-K and /F(NO 2)-E-A-Nle-S, indicates that the P2′ residue is highly constrained, while the positioning of the P1′ and P3′ residues are sequence dependent.
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, 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 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.
Proceedings of the National Academy of Sciences, 1990
The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor Abbreviations: HIV-1, human immunodeficiency virus 1; HEA, hydroxyethylamine. tTo whom reprint requests should be addressed. 'The atomic coordinates have been deposited in the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (reference 7HVP). 8805 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.