Structure of Complex of Synthetic HIV-1 Protease with a Substrate-Based Inhibitor at 2.3 Å Resolution

Miller, Maria; Schneider, Jens; Sathyanarayana, Bangalore K.; Tóth, Máté; Marshall, Garland R.; Clawson, Leigh; Selk, Linda; Kent, Stephen B. H.; Wlodawer, Alexander

doi:10.1126/science.2686029

Cited by 682 publications

(435 citation statements)

References 17 publications

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“…In the two cocrystal structures of HIV-l PR [4,5], the inhibitor, as proposed from a model complex [7], binds in an extended P-conformation with similar interactions to those observed in the structures of complexes of nonviral aspartic proteases with inhibitors [ 13,16-201. There are hydrogen bond interactions to the inhibitor provided by residues near the two catalytic triplets, and by residues from the flap (residues 74 to 76 in pepsin).…”

Section: Resultsmentioning

confidence: 99%

“…The structure of HIV-l PR is PDB entry 3HVP [2]. The complex of HIV-l PR with reduced peptide inhibitor, MVT-101, (Ac-Thr-Ile-Nle-t[CHz-NH]-Nle-Gln-Arg-amide, where Nle is norleucine and Ki = 780 nM) [4] is PDB 4HVP, while the coordinates of a complex with inhibitor JG-365 (Ac-Ser-Leu-Ans-Phe-…”

Section: Methodsmentioning

confidence: 99%

“…More recently, the structure of HIV-l PR complexed with an inhibitor has been determined [4] and a second cocrystal structure with a different inhibitor is available [5]. The catalytically active dimer of retroviral PR is structurally similar to the monomeric nonviral aspartic proteases which consist of two domains.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of inhibitor binding in HIV‐1 protease and in non‐viral aspartic proteases: the role of the flap

Gustchina

Weber

1990

FEBS Letters

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The crystal structure of HIV-l protease with an inhibitor has been compared with the structures of non-viral aspartic proteases complexed with inhibitors. In the dimeric HIV-l protease, two 4-stranded /?-sheets are formed by half of the inhibitor, residues 27-29, and the flap from each monomer. In the monomeric non-viral enzyme the single flap does not form a/I-sheet with an inhibitor. The HIV-l protease shows more interactions with a longer peptide inhibitor than are observed in non-viral aspartic protease-inhibitor complexes. This, and the large movement of the flaps, restricts the conformation of the protease cleavage sites in the retroviral polyprotein precursor.Retroviral protease; Aspartic protease; Enzyme-substrate interaction; Retroviral polyprotein precursor

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Comparison of inhibitor binding in HIV‐1 protease and in non‐viral aspartic proteases: the role of the flap

Gustchina

Weber

1990

FEBS Letters

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“…The substrate binding cleft and the catalytic aspartic residues are located between these lobes (for review, see Davies [1990]). On the other hand, the retroviral aspartic proteases, including the HIV protease, are homodimers with the substrate binding cleft and the active-site aspartic residues equally contributed by the two monomers (Foundling et al, 1989;Lapatto et al, 1989;Miller et al, 1989;Wlodawer et al, 1383Fitzgerald et al, 1990). It has been proposed that the internally homologous lobes of pepsin and other eukaryotic aspartic proteases are evolutionarily derived by gene duplication and fusion, and that a primordial aspartic protease would have a homodimeric structure (Tang et al, 1978).…”

Section: ~ ~~~~mentioning

confidence: 99%

Conformational instability of the N‐ and C‐terminal lobes of porcine pepsin in neutral and alkaline solutions

et al. 1993

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Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two-chain pepsin or a two-chain pepsinogen (Lin, X . , et al., 1992, J. Biol. Chem. 267,[17257][17258][17259][17260][17261][17262][17263]. Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline-inactivated pepsin was regenerated by the addition of the recombinant N-terminal lobe but not by the C-terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N-terminal lobe. A complex between recombinant N-terminal lobe of pepsinogen and alkaline-denatured pepsin has been isolated. This complex is structurally similar to a two-chain pepsinogen, but it contains an extension of a denatured pepsin N-terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N-terminal lobe. The structural components that are responsible for the alkaline instability of the N-terminal lobe are likely to be carboxyl groups with abnormally high pK, values. The electrostatic potentials of 23 net carboxyl groups in the N-terminal domain (as compared to 19 in the C-terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp", Asp"', Glu4, Glu13, and Asp"'. Especially, the partially buried Asp", which interacts with Asp159, could cause one of these two groups to have an abnormally high pK, and the other an abnormally low pK, value. Thus, the ionization of Asp'' at a high pH may place two negatively charged residues in close vicinity. This unfavorable situation may be the trigger for the denaturation of the N-terminal lobe of pepsin.Keywords: carboxyl ionization; denaturation; domain structure; electrostatic potentials; porcine pepsinThe fact that some aspartic proteases, such as HIV protease and renin, are involved in human diseases has stimulated much interest recently in the understanding of the structure-function relationships of this group of enzymes. Reprint requests to: Jordan Tang, Protein Studies Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, Oklahoma 73104.Abbreviations: bis-Tris, bis(2-hydroxyethyl)-iminotris(hydroxymethy1)methane; FPLC, fast protein liquid chromatography (chromatograph); HIV, human immunodeficiency virus; PAGE, polyacrylamide gel electrophoresis; pep, the NH,-terminal lobe of pepsin; pep.sin, twochain pepsin; pepsin,, alkaline-inactivated pepsin; pro, propeptide; propep, the NH2-terminal lobe of pepsinogen; propep.pepsin,, complex between propep and alkaline-inactivated pepsin; propep.sin, twochain pepsinogen; SDS, sodium dodecyl sulfate; sin, the C-terminal lob...

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“…Rao, unpubl.). That model in turn was originally based on the structure of Miller et al (1989), refined further at 2 A resolution. The very first (IF,[ -IFCl)ac map using the phases calculated from the protein alone clearly showed the inhibitor density in the active site cleft.…”

Section: Structure Solution and Refinementmentioning

confidence: 99%

Crystal structure of a complex of HIV‐1 protease with a dihydroxyethylene‐containing inhibitor: Comparisons with molecular modeling

Thanki¹,

Rao²,

Foundling³

et al. 1992

Protein Science

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The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV-1) protease with a peptide-mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been de- The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X-ray structure with a model-built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side-chain atoms in the P2-P2' region, where the interaction with the protein is strongest. However, the X-ray structure and the model differ considerably in the location of the P3 and P3' end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a PI-PI' geometry very close to that seen in the X-ray structure.

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Structure of Complex of Synthetic HIV-1 Protease with a Substrate-Based Inhibitor at 2.3 Å Resolution

Cited by 682 publications

References 17 publications

Comparison of inhibitor binding in HIV‐1 protease and in non‐viral aspartic proteases: the role of the flap

Comparison of inhibitor binding in HIV‐1 protease and in non‐viral aspartic proteases: the role of the flap

Conformational instability of the N‐ and C‐terminal lobes of porcine pepsin in neutral and alkaline solutions

Crystal structure of a complex of HIV‐1 protease with a dihydroxyethylene‐containing inhibitor: Comparisons with molecular modeling

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