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1.
Kinetic studies of human immunodeficiency virus type 1 protease and its active-site hydrogen bond mutant A28S. 总被引:13,自引:0,他引:13
Human immunodeficiency virus type 1 (HIV-1) protease optimally catalyzes in the pH range of 4-6 in contrast to nearly all of the other eukaryotic aspartic proteases, which catalyze best in the pH range of 2-4. A possible structural reason for the higher optimal pH of HIV-1 protease is the absence of a hydrogen bond to the carboxyl group of active-site Asp25, which is nearly universally present in others. To investigate this hypothesis, we have mutated residue 28 in HIV-1 protease from alanine to serine. Both the wild-type and the mutant A28S enzymes have been overexpressed in Escherichia coli using a chemically synthesized gene and purified for a comparative study in enzyme kinetics. The kcat and Km values were determined by a radiometric assay for the wild-type enzyme from pH 3.2 to 7.0, and for the mutant enzyme from pH 3.2 to 6.0. The low pK values of the active site of the free enzyme, pKe1, are 3.3 and 3.4 for the wild-type and mutant enzymes, respectively. The low pK values of the active site of the enzyme bound to substrate, pKes1, are 5.1 and 4.3 for the wild-type and mutant enzymes, respectively. The high pK values of the free enzyme, pKe2, are 6.8 and 5.6, and the corresponding ones for the substrate-bound enzyme, pKes2, are 6.9 and 6.0 for the wild-type and mutant enzymes, respectively. The lowering of pK values in mutant HIV-1 protease indicates that the hydroxyl group of Ser28 forms a new hydrogen bond to active-site Asp25 to increase its acidity. 相似文献
2.
Crystal structure of an in vivo HIV-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance
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Hong L Zhang XC Hartsuck JA Tang J 《Protein science : a publication of the Protein Society》2000,9(10):1898-1904
Saquinavir is a widely used HIV-1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV-1 viral enzymes. Three HIV-1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5-, 3-, and 419-fold increase in Ki values, respectively, compared to the wild-type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364-12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV-1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 A resolution. This mutant complex is compared with that of the wild-type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340-3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main-chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1' substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild-type enzyme and therefore may facilitate catalysis by the resistant mutant. 相似文献
3.
Human immunodeficiency virus type-1 reverse transcriptase and ribonuclease h as substrates of the viral protease
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Alfredo G. Tomasselli Jean L. Sarcich Linda J. Barrett Ilene M. Reardon W. Jeffrey Howe David B. Evans Satish K. Sharma Robert L. Heinrikson 《Protein science : a publication of the Protein Society》1993,2(12):2167-2176
4.
A highly selective and facile assay of human immunodeficiency virus protease (HIV-PR) has been required for the screening of medicinal inhibitors and also for classifying the subtypes of HIV in the therapeutic treatment of acquired immune deficiency syndrome (AIDS). This article describes a novel assay method of HIV-PR based on the selective fluorogenic reaction of peptides. A peptide fragment generated from a substrate by the enzymatic digestion with HIV-PR could be selectively quantified by the spectrofluorometric detection after the fluorogenic reaction with catechol in the presence of sodium periodate and sodium borate (pH 7.0). This assay system uses an N-terminal acetyl peptide as the substrate and crude extracts from Escherichia coli expressing recombinant HIV-PR. The activity obtained by the proposed assay correlated with that obtained by a conventional HIV-PR assay based on fluorescence resonance energy transfer detection. 相似文献
5.
Under the selection pressure of drugs, mutations appear in HIV-1 protease even at the sites, which are conserved in the untreated individuals. Cysteine 95 is a highly conserved residue and is believed to be involved in regulation of HIV-1 protease. In some of the virus isolates from patients undergoing heavy treatment with anti-HIV protease drugs, C95F mutation has appeared. The present study reports 1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin. It is found that in this mutant, dimer interface has become more rigid and that the packing at the interface of terminal and core domains is altered. These alterations may be relevant to C95F mutation conferring drug resistance to HIV-1 protease. 相似文献
6.
X-ray crystal structures of human immunodeficiency virus type 1 protease mutants complexed with atazanavir
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Klei HE Kish K Lin PF Guo Q Friborg J Rose RE Zhang Y Goldfarb V Langley DR Wittekind M Sheriff S 《Journal of virology》2007,81(17):9525-9535
Atazanavir, which is marketed as REYATAZ, is the first human immunodeficiency virus type 1 (HIV-1) protease inhibitor approved for once-daily administration. As previously reported, atazanavir offers improved inhibitory profiles against several common variants of HIV-1 protease over those of the other peptidomimetic inhibitors currently on the market. This work describes the X-ray crystal structures of complexes of atazanavir with two HIV-1 protease variants, namely, (i) an enzyme optimized for resistance to autolysis and oxidation, referred to as the cleavage-resistant mutant (CRM); and (ii) the M46I/V82F/I84V/L90M mutant of the CRM enzyme, which is resistant to all approved HIV-1 protease inhibitors, referred to as the inhibitor-resistant mutant. In these two complexes, atazanavir adopts distinct bound conformations in response to the V82F substitution, which may explain why this substitution, at least in isolation, has yet to be selected in vitro or in the clinic. Because of its nearly symmetrical chemical structure, atazanavir is able to make several analogous contacts with each monomer of the biological dimer. 相似文献
7.
Triton X-100-extracted human skin fibroblasts were exposed to human immunodeficiency virus type 1 protease and analysed by 2D-gel electrophoresis and immunofluorescence microscopy. Vimentin, two of the tropomyosin isoforms, a protein with Mr ∼ 90,000 and a protein with Mr ∼ 200,000 were found to be degraded. Structurally, this was accompanied by the disintegration of the vimentin filament network and the disappearance of the microfilament network. In contrast to our in vivo observations (Höner et al., 1991), prominent stress fibers and chromatin structure seemed to be rather resistant to the action of this protease. 相似文献
8.
9.
Active-site residues of cyclophilin A are crucial for its incorporation into human immunodeficiency virus type 1 virions. 总被引:4,自引:4,他引:0
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Human immunodeficiency virus type 1 (HIV-1) incorporates the cellular peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). CsA inhibits the incorporation of CyPA and reduces HIV-1 virion infectivity but is inactive against closely related primate lentiviruses that do not interact with CyPA. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction with a proline-containing, solvent-exposed loop in the capsid (CA) domain of the Gag polyprotein. CsA, which disrupts the interaction with CA, binds at the active site of CyPA. To test whether active-site residues are also involved in the interaction with HIV-1 CA, we used a panel of previously characterized active-site mutants of human CyPA. Expression vectors for epitope-tagged wild-type and mutant CyPA were transfected into COS-gamma cells along with HIV-1 proviral DNA, and the virions produced were analyzed for the presence of tagged proteins. Cotransfection of the wild-type expression vector led to the incorporation of readily detectable amounts of epitope-tagged CyPA into HIV-1 virions. One CyPA mutant with a substantially decreased sensitivity to CsA was incorporated with wild-type efficiency, demonstrating that the requirements for binding to CsA and to HIV-1 CA are not identical. The remaining six CyPA mutants were incorporated with markedly reduced efficiency, providing in vivo evidence that HIV-1 CA interacts with the active site of CyPA. 相似文献
10.
Based on the assumption that fluidity of the plasma membrane and viral envelope is necessary for recruiting additional receptors and ligands to the initial attachment site for "multiple-site binding," we determined the effect of increased temperature on viral infectivity. Infection of human immunodeficiency virus type 1 (HIV-1) and a pseudotyped luciferase-expressing chimeric virus using MAGI and GHOST/CXCR4 cells showed that in 1 hr of viral adsorption the extent of virus infection and the amount of tightly adsorbed viruses depended on temperature; and that membrane fluidity increased according to increased temperature. Augmented infection was observed as post-attachment enhancement (PAE) when cells were washed and incubated at 40 C for 1 hr after viral adsorption. PAE was completely inhibited by 1 micro M of anti-CXCR4 peptide T140, and addition of T140 at 20 min resulted in a gradual loss of inhibition of PAE, indicating the need for a 30 to 40 min timelag to ensure tight multiple-site binding. These data suggest that the accumulation of gp120 and receptor complex (multiple-site binding) was needed to complete the infection. Treatments of cells with 0.05% Tween 20 or 2 micro g/ml of anti-HLA-II antibody resulted in increases or decreases, respectively, of attached viruses and the infectivity. As well, Tween 20 and anti-HLAII antibody enhanced and suppressed the fluidity of the plasma membrane, respectively. Amounts of adsorbed viruses and degrees of viral infectivity correlated with the intensity of fluidity of the plasma membrane, probably due to the formation of multiple-site binding. 相似文献
11.
12.
A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir
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Ziermann R Limoli K Das K Arnold E Petropoulos CJ Parkin NT 《Journal of virology》2000,74(9):4414-4419
Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs-saquinavir, indinavir, ritonavir, and nelfinavir-has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5- to 12. 5-fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy. 相似文献
13.
Factor analysis scales of generalized amino acid information (FASGAI) involving hydrophobicity, alpha and turn propensities, bulky properties, compositional characteristics, local flexibility, and electronic properties were derived from 516 property parameters of 20-coded amino acids, and was then employed to represent sequence structures of 746 peptides with 8 amino acid residues. Cleavage site prediction models for human immunodeficiency virus type 1 protease by linear discriminant analysis and support vector machine with radial basis function kernel were constructed to identify if they could be cleaved or not, and were further utilized to investigate the cleavage specificity. These diversified properties, including the bulky properties, secondary conformation characteristics, electronic properties, and hydrophobicity at the first, the second, the fourth, the fifth, and the sixth residue, are possibly important factors in determining HIV PR cleavage or not. Particularly, maximal positive and negative influences result from the bulky properties of different sites. Further results from analysis of variance also likely reflect that the HIV PR recognizes diversified key properties of various sites in the octameric sequences. Satisfactory results show that FASGAI can not only be used to represent sequence structures of various functional peptides, but alsoprovide a potential feasible measure for exploring relationship between protein motif sequences and their functions. 相似文献
14.
Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity
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Foulkes JE Prabu-Jeyabalan M Cooper D Henderson GJ Harris J Swanstrom R Schiffer CA 《Journal of virology》2006,80(14):6906-6916
Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain. 相似文献
15.
Liu F Kovalevsky AY Louis JM Boross PI Wang YF Harrison RW Weber IT 《Journal of molecular biology》2006,358(5):1191-1199
Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR(F53L) showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR(F53L) was determined in the unliganded form at 1.35 Angstrom resolution in space group P4(1)2(1)2. The tips of the flaps in PR(F53L) had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50'. The changes in interactions between the flaps agreed with the reduced stability of PR(F53L) relative to wild-type PR. The altered flap interactions in the unliganded form of PR(F53L) suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants. 相似文献
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18.
Kovalevsky AY Liu F Leshchenko S Ghosh AK Louis JM Harrison RW Weber IT 《Journal of molecular biology》2006,363(1):161-173
TMC114 (darunavir) is a promising clinical inhibitor of HIV-1 protease (PR) for treatment of drug resistant HIV/AIDS. We report the ultra-high 0.84 A resolution crystal structure of the TMC114 complex with PR containing the drug-resistant mutation V32I (PR(V32I)), and the 1.22 A resolution structure of a complex with PR(M46L). These structures show TMC114 bound at two distinct sites, one in the active-site cavity and the second on the surface of one of the flexible flaps in the PR dimer. Remarkably, TMC114 binds at these two sites simultaneously in two diastereomers related by inversion of the sulfonamide nitrogen. Moreover, the flap site is shaped to accommodate the diastereomer with the S-enantiomeric nitrogen rather than the one with the R-enantiomeric nitrogen. The existence of the second binding site and two diastereomers suggest a mechanism for the high effectiveness of TMC114 on drug-resistant HIV and the potential design of new inhibitors. 相似文献
19.
The production of alkaline protease from Thermoactinomyces sp. E79 was repressed by 0.2% (w/v) glucose in the medium. Catabolite repression-resistant mutant M1 was obtained by combined treatment of UV light and N-methyl- N-nitro-N-nitrosoguanidine. The glucose uptake studies accomplished by [14C]glucose showed that the mutant has lost its ability for glucose uptake. The protease production by mutant M1 in the enzyme production medium was 62 U/mg, which was twice that of the wild-type strain. 相似文献
20.
Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant
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Ott DE Coren LV Chertova EN Gagliardi TD Nagashima K Sowder RC Poon DT Gorelick RJ 《Journal of virology》2003,77(10):5547-5556
The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10(-6) relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC. 相似文献