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Beltz H Clauss C Piémont E Ficheux D Gorelick RJ Roques B Gabus C Darlix JL de Rocquigny H Mély Y 《Journal of molecular biology》2005,348(5):1113-1126
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Stephen AG Worthy KM Towler E Mikovits JA Sei S Roberts P Yang QE Akee RK Klausmeyer P McCloud TG Henderson L Rein A Covell DG Currens M Shoemaker RH Fisher RJ 《Biochemical and biophysical research communications》2002,296(5):1228-1237
The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of approximately 2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4('),5('),6(')-trihydroxy-3(')-oxo-3H-xanthen-9(')-yl)benzoic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC(50) of 16.6+/-4.3 microM (means+/-SD). Synthetic variants lacking the two hydroxyls at positions 4(') and 5(') in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33). 相似文献
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Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1. 总被引:26,自引:4,他引:26 下载免费PDF全文
M. F. Summers L. E. Henderson M. R. Chance J. W. Bess Jr T. L. South P. R. Blake I. Sagi G. Perez-Alvarado R. C. Sowder rd D. R. Hare et al. 《Protein science : a publication of the Protein Society》1992,1(5):563-574
All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS. 相似文献
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Cruceanu M Stephen AG Beuning PJ Gorelick RJ Fisher RJ Williams MC 《Analytical biochemistry》2006,358(2):159-170
We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single lambda-DNA molecules through the helix-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7's capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. 相似文献
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The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1 总被引:12,自引:0,他引:12
Gabus C Derrington E Leblanc P Chnaiderman J Dormont D Swietnicki W Morillas M Surewicz WK Marc D Nandi P Darlix JL 《The Journal of biological chemistry》2001,276(22):19301-19309
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A unimolecular oligonucleotide switch, termed here an AlloSwitch, binds the mature HIV-1 nucleocapsid protein, NCp7. This switch can be used as an indicator for the presence of free NCp7 and NC domains in precursor and fusion proteins. It is thermodynamically stable in two conformations, H and O. A FRET pair is covalently attached to the strands to report on the molecular state of the switch. The results show that NC has an affinity for O 170 times higher than its affinity for H and that in the absence of NC the equilibrium ratio K1 = [O]/[H] = 0.10 +/- 0.03 for the switch sequence reported here. The change between the two states happens on a rapid kinetic time scale. A framework is introduced to aid in the design of AlloSwitches aimed at other targets. A high-affinity probe segment must be available to bind the target in the O-form, while a cover segment hides the probe in H. A key is adjusting the cover sequence to favor the H-form by a factor of 10-1000. This affords a robust response to small changes in target concentration, while saturation produces more than 90% of the maximal change in fluorescence. When a competitor displaces the switch from the NC-O complex, the released switch reverts to the H-form. This is the basis for a mix-and-read strategy for high-throughput screening of anti-nucleocapsid drug candidates that is much simpler to execute than traditional assays that require immobilization and washing steps. 相似文献
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Matthew J. Belousoff Gilles Gasser Bim Graham Yitzhak Tor Leone Spiccia 《Journal of biological inorganic chemistry》2009,14(2):287-300
A peptide nucleic acid (PNA) oligomer and a series of PNA conjugates featuring covalently attached pendant 1,4,7,10-tetraazacyclododecane
(cyclen) or bis((pyridin-2-yl)methyl)amine (DPA) moieties have been synthesized that are complementary to regions of the HIV-1
TAR messenger RNA stem-loop. Thermal denaturation studies, in conjunction win with native gel shift assays, suggest that the
PNAs “invade” TAR to produce a mixture of two 1:1 PNA–TAR adducts, tentatively assigned as an “open-duplex” structure, in
which the TAR stem-loop dissociates and the PNA hybridizes with its RNA complement via Watson–Crick base-pairing, and a triplex-type
structure, in which the initially displaced RNA segment is bound to the PNA:RNA duplex through Hoogsteen base-pairing. Thermal
denaturation experiments with the TAR sequence and single-stranded RNA and DNA oligonucleotides, both in the presence and
in the absence of Zn2+ ions, show that the introduction of cyclen or DPA ligand arms into the PNA oligomer leads to a small but reproducible increase
in the T
m values. This is attributed to hydrogen-bonding and/or electrostatic interactions between protonated forms of cyclen/DPA and
the cognate RNA or DNA oligonucleotide targets. Contrary to expectations, the addition of Zn2+ ions did not further enhance duplex formation through binding of Zn(II)–cyclen or Zn(II)–DPA moieties to the complementary
RNA or DNA. Native gel shift assays further confirmed the stability increase of the metal-free cyclen- and DPA-modified PNA
hybrids as compared with a control PNA sequence.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Selection and stabilization of the RNA aptamers against the human immunodeficiency virus type-1 nucleocapsid protein 总被引:5,自引:0,他引:5
Kim SJ Kim MY Lee JH You JC Jeong S 《Biochemical and biophysical research communications》2002,291(4):925-931
The nucleocapsid (NC) protein of the human immunodeficiency virus-1 (HIV-1) plays an important role in the encapsidation of viral RNA and assembly of viral particle. Since the NC protein is resistant for mutation, it might be an excellent target for the anti-viral therapy. RNA aptamers that bind to the mature form of the NC protein were isolated from a RNA library. Surface plasmon resonance measurement and gel shift assay showed that the RNA aptamers specifically bind to the NC protein with high affinity and compete for the psi RNA binding to the NC protein. Mapping of the RNA aptamer showed at least two sites for the protein binding, suggesting a multiple and cooperative binding by the NC to RNA. In addition, the circular form of RNA avidly binds to the NC protein as a linear counter does. Stabilized RNA aptamer is expected to act as an inhibitor for the viral packaging. 相似文献
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