Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.
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PMID:Ordered processing of the human immunodeficiency virus type 1 GagPol precursor is influenced by the context of the embedded viral protease. 1605 52

Topological scores, measures of sequence-structure compatibility, are calculated for all 1,881 single point mutants of the human immunodeficiency virus (HIV)-1 protease using a four-body statistical potential function based on Delaunay tessellation of protein structure. Comparison of the mutant topological score data with experimental data from alanine scan studies specifically on the dimer interface residues supports previous findings that 1) L97 and F99 contribute greatly to the Gibbs energy of HIV-1 protease dimerization, 2) Q2 and T4 contribute the least toward the Gibbs energy, and 3) C-terminal residues are more sensitive to mutations than those at the N-terminus. For a more comprehensive treatment of the relationship between protease structure and function, mutant topological scores are compared with the activity levels for a set of 536 experimentally synthesized protease mutants, and a significant correlation is observed. Finally, this structure-function correlation is similarly identified by examining model systems consisting of 2,015 single point mutants of bacteriophage T4 lysozyme as well as 366 single point mutants of HIV-1 reverse transcriptase and is hypothesized to be a property generally applicable to all proteins.
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PMID:Computational mutagenesis studies of protein structure-function correlations. 1661 25

The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe-Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 degrees C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.
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PMID:Isolation and characterization of recombinant Drosophila Copia aspartic proteinase. 1681 67

Protein-protein and protein-ligand interactions play a central role in biochemical reactions, and understanding these processes is an important task in different fields of biomedical science and drug discovery. Proteins often work in complex assemblies of several macromolecules and small ligands. The structural and functional description of protein-protein interactions (PPI) is very important for basic-, as well as applied research. The interface areas of protein complexes have unique structure and properties, so PPI represent prospective targets for a new generation of drugs. One of the key targets of PPI inhibitors are oligomeric enzymes. This report shows interactive links between virtual and experimental approaches in a total pipeline "from gene to drug" and using Surface Plasmon Resonance technology for experimentally assessing PPI. Our research is conducted on two oligomeric enzymes -- HIV-1 protease (HIVp) (homo-dimer) and bacterial L-asparaginase (homo-tetramer). Using methods of molecular modeling and computational alanine scanning we obtained structural and functional description of PPI in these two enzymes. We also presented a real example of application of integral approach in searching inhibitors of HIVp dimerization -- from virtual database mining up to experimental testing of lead compounds.
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PMID:Protein-protein interactions as new targets for drug design: virtual and experimental approaches. 1763 63

As part of our ongoing studies of the human immunodeficiency virus type 1 (HIV-1) protease enzyme, we set out to develop a modular chemical synthesis of the protein from multiple peptide segments. Our initial attempts were frustrated by the insolubility of intermediate peptide products. To overcome this problem, we designed a synthetic strategy combining the solubility-enhancing properties of C-terminal (Arg)n tags and the biological phenomenon of autoprocessing of the Gag-Pol polyprotein that occurs during maturation of the HIV-1 virus in vivo. Synthesis of a 119-residue peptide chain containing 10 residues of the reverse transcriptase (RT) open reading frame plus an (Arg)(10) tag at the C-terminus was straightforward by native chemical ligation followed by conversion of the Cys residues to Ala by Raney nickel desulfurization. The product polypeptide itself completed the final synthetic step by removing the C-terminal modification under folding conditions, to give the mature 99-residue polypeptide. High-purity homodimeric HIV-1 protease protein was obtained in excellent yield and had full enzymatic activity; the structure of the synthetic enzyme was confirmed by X-ray crystallography to a resolution of 1.07 A. This efficient modular synthesis by a biomimetic autoprocessing strategy will enable the facile synthesis of unique chemical analogues of the HIV-1 protease to further elucidate the molecular basis of enzyme catalysis.
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PMID:Modular total chemical synthesis of a human immunodeficiency virus type 1 protease. 1770 84

HIV-1 protease (PR) mediates the proteolytic processing of virus particles during or after virus budding. PR activation is thought to be triggered by appropriate Gag-Pol/Gag-Pol interaction; factors affecting this interaction either enhance or reduce PR-mediated cleavage efficiency, resulting in markedly reduced virion production or the release of inadequately processed virions. We previously showed that a Gag-Pol deletion mutation involving the reverse transcriptase tryptophan (Trp) repeat motif markedly impairs PR-mediated virus maturation and that an alanine substitution at W401 (W401A) or at both W401 and W402 (W401A/W402A) partially or almost completely negates the enhancement effect of efavirenz (a nonnucleoside reverse transcriptase inhibitor) on PR-mediated virus processing efficiency. These data suggest that the Trp repeat motif may contribute to the PR activation process. Here we demonstrate that due to enhanced Gag cleavage efficiency, W402 alanine or leucine substitution significantly reduces virus production. However, W402 replacement with phenylalanine does not significantly affect virus particle assembly or processing, but it does markedly impair viral infectivity in a single-cycle infection assay. Our results demonstrate that a single amino acid substitution at HIV-1 RT can radically affect virus assembly by enhancing Gag cleavage efficiency, suggesting that in addition to contributing to RT biological function during the early stages of virus replication, the HIV-1 RT tryptophan repeat motif in a Gag-Pol context may play an important role in suppressing the premature activation of PR during late-stage virus replication.
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PMID:A single amino acid substitution in HIV-1 reverse transcriptase significantly reduces virion release. 1988 67

The structural and functional role of conserved residue G86 in HIV-1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PR(G86A) and PR(G86S)). While PR(G86S) had undetectable catalytic activity, PR(G86A) exhibited approximately 6000-fold lower catalytic activity than PR. (1)H-(15)N NMR correlation spectra revealed that PR(G86A) and PR(G86S) are dimeric, exhibiting dimer dissociation constants (K(d)) of approximately 0.5 and approximately 3.2 muM, respectively, which are significantly lower than that seen for PR with R87K mutation (K(d) > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PR(G86A) and PR(G86S) in the presence of active-site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. (c) 2009 Wiley-Liss, Inc.
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PMID:Highly conserved glycine 86 and arginine 87 residues contribute differently to the structure and activity of the mature HIV-1 protease. 1989 62

HNN has proven to be an extremely valuable experiment for rapid and unambiguous backbone (H(N), (15)N) assignment in ((13)C, (15)N) labeled proteins. However, low sensitivity of the experiment is often a limiting factor, especially when the transverse relaxation times (T(2)) are short. We show here that BEST modification Schanda et al. (2006) [2] increases the sensitivity per unit time by more than a factor of 2.0 and thus substantially increases the speed of data collection; good 3D data can be collected in 8-10h. Next, we present a simple method for amino-acid type identification based on simple 2D versions of the HNN experiment, labeled here as 2D-(HN)NH. Each of these experiments which produce anchor points for Gly, Ala, Ser/Thr residues, can be recorded in less than an hour. These enable rapid data acquisition, rapid analysis, and consequently rapid assignment of backbone (H(N), (15)N) resonances. The 2D-(HN)NH experiment does not involve aliphatic/aromatic protons and hence can be applied to deuterated protein samples as well, which is an additional advantage. The experiments have been demonstrated with human ubiquitin (76 aa) and acetic-acid denatured HIV-1 protease (99 aa), as representatives of folded and unfolded protein systems, respectively.
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PMID:BEST-HNN and 2D-(HN)NH experiments for rapid backbone assignment in proteins. 2023 46

HIV-1 protease is a very attractive target for the development of new anti-HIV drugs and has been extensively studied over the past decades. In this study, we present a detailed atomic level characterization of the dimer interface in the enzyme HIV-1 protease through computational alanine scanning mutagenesis and molecular dynamics simulations. In addition to a full mapping of the amino acid residues present at the subunit interface, in terms of the corresponding energetic contribution for dimer formation and of their classification as hot spots, warm spots, and null spots, we trace a dynamic analysis of the subunit interacting and solvent accessible surface areas and of the most important hydrogen bonds between subunits. The results presented illustrate the high energetic importance for dimer formation of a small set of five amino acid residue pairs at the subunit interface-Leu5, Ile50, Arg87, Leu97, and Phe99-and provide important clues on the most important structural and energetic determinants for dimer formation. In addition, the results presented suggest several key targets at the subunit interface for the development of new molecules that aim to inhibit HIV-1 protease (PR) activity through blocking the formation of the fully active PR homodimeric form, providing important clues for drug design.
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PMID:Detailed atomistic analysis of the HIV-1 protease interface. 2154 27

The binding properties of the protein-inhibitor complex of human immunodeficiency virus type 1 (HIV-1) protease with the inhibitor TMC-126 are investigated by combining computational alanine scanning (CAS) mutagenesis with binding free-energy decomposition (BFED). The calculated results demonstrate that the flap region (residues 38-58) and the active site region (residues 23-32) in HIV-1 protease contribute 63.72% of the protease to the binding of the inhibitor. In particular, the mechanisms for the interactions of key residues of these species are fully explored and analyzed. Interestingly, the regression analyses show that both CAS and BFED based on the generalized Born model yield similar results, with a correlation coefficient of 0.94. However, compared to CAS, BFED is faster and can decompose the per-residue binding free-energy contributions into backbone and side-chain contributions. The results obtained in this study are useful for studying the binding mechanism between receptor and ligand and for designing potent inhibitors that can combat diseases.
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PMID:Insights into the structural function of the complex of HIV-1 protease with TMC-126: molecular dynamics simulations and free-energy calculations. 2185 May 70


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