Preliminary investigation of the interaction between the R2R3 DNA binding domain of the oncoprotein c‐Myb and DNA fragments

Jamin, Nadège; Tilly, Véronique Le; Zargarian, Loussiné; Bostad, Anne; Besançon‐Yoshpe, Iris; Lirsac, Pierre-Noël; Gabrielsen, Odd S.; Toma, Flavio

doi:10.1002/(sici)1097-461x(1996)59:4<333::aid-qua7>3.3.co;2-7

Cited by 3 publications

(3 citation statements)

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“…The affinity of rhERα for EREcs and rt ERE sequences was determined by using fluorescence anisotropy methodology. Fluorescence anisotropy DNA binding curve has been already used for determining protein−DNA equilibrium ( − ). Previous studies performed at low salt concentration have reported that affinity of rhERα for ERE, either consensus or mutated motif with a single base-pair change per half-site, is roughly the same ( K D ≈ 2 nM) with the same rhERα/ERE stoichiometry ().…”

Section: Resultsmentioning

confidence: 99%

Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

2004

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Previously, we have identified an imperfect estrogen response element (rtERE) in the promoter of the rainbow trout vitellogenin gene. Although this ERE leads to a lower transcriptional activation, a better estradiol stimulation in vivo as compared to consensus ERE (EREcs) was observed. Here we examine the ability of recombinant human estrogen receptor alpha (rhERalpha) to bind DNA containing the EREcs or the natural imperfect rtERE, which contains three mismatches. At low salt concentration, whatever the ERE sequence, dissociation equilibrium constants of the specific rhERalpha-ERE complexes are similar (K(D) = 2 nM) with the same stoichiometry. As salt concentration increases from 80 to 200 mM KCl, the affinity of the rhERalpha-rtERE complex largely diminishes whereas that of rhERalpha-EREcs seems less affected. Hence the nature of the interactions stabilizing these complexes is different: more ionic in rhERalpha-rtERE as compared to rhERalpha-EREcs. Moreover, kinetic measurements showed that specific rhERalpha-ERE complexes exhibit shorter half-lives (few seconds) and that the rhERalpha-EREcs complex is more stable (33 s) than the complex that formed with rtERE (19.8 s), in accordance with equilibrium binding results. Finally, dynamic studies of rhERalpha have shown that the protein fluctuations are damped when the salt concentration increases or when bound to ERE and all the more with rtERE. The interplay of affinity, complex half-lives, and protein dynamics in the transcriptional regulation of estrogen receptor is discussed.

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Section: Resultsmentioning

confidence: 99%

Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

2004

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“…For instance, in their pioneering work using fluorescence anisotropy, Le Tilly and Royer (68), have found a K d of 0.13 nM for the binding of the trp repressor protein to its specific binding sites. A high affinity has been also obtained for the interaction between DNA and the DNA binding domain of c-Myb, with K d of ϳ5 nM (69). In such complexes the number of contacts between the protein and the DNA are not so numerous, hardly more than those exchanged between an oligopeptide and its nucleic acid site; nevertheless, the affinities achieved are considerable.…”

Section: Figmentioning

confidence: 94%

Strategy to Discriminate between High and Low Affinity Bindings of Human Immunodeficiency Virus, Type 1 Integrase to Viral DNA

Zargarian

Benleumi

Renisio

et al. 2003

Journal of Biological Chemistry

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The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix ␣4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the ␣4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (K d of 1.5 nM). Data corroborate previous in vivo results obtained with mutated viruses.

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“…Recently, two groups have reported the results of NMR studies of c-MybR2R3-DNA complexes (25)(26)(27)44). In a complex formed with a target site based on the mim-1-A Myb binding site (GTAACGGTCTAC), the imino proton resonances of base pairs 3, 4, 7, and 8 (with the core TAACGG sequence corresponding to base pairs 1-6) were found to be doubled with a roughly 60:40 intensity ratio, suggesting the presence of two different forms of the complex in slow exchange (44). These effects are strikingly similar to those seen for a number of the protein signals in spectra of B-MybR2R3 bound to the tom-1-A site (TCCTTAACG-GACTGAG).…”

Section: Solution Structure Of the B-myb Dna-binding Domainmentioning

confidence: 99%

Solution Structure of the B-Myb DNA-Binding Domain: A Possible Role for Conformational Instability of the Protein in DNA Binding and Control of Gene Expression^,

et al. 1998

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Double- and triple-resonance heteronuclear NMR spectroscopy have been used to determine the high-resolution solution structure of the minimal B-Myb DNA-binding domain (B-MybR2R3) and to characterize the specific complex formed with a synthetic DNA fragment corresponding to the Myb target site on the Myb-regulated gene tom-1. B-MybR2R3 is shown to consist of two independent protein domains (R2 and R3) joined by a short linker, which have strikingly different tertiary structures despite significant sequence similarities. In addition, the C-terminal region of B-Myb R2 is confirmed to have a poorly defined structure, reflecting the existence of multiple conformations in slow to intermediate exchange. This contrasts with the tertiary structure reported for c-MybR2R3, in which both R2 and R3 have the same fold and the C-terminal region of R2 forms a stable, well-defined helix [Ogata, K., et al. (1995) Nat. Struct. Biol. 2, 309-320]. The NMR data suggest there are extensive contacts between B-MybR2R3 and its DNA target site in the complex and are consistent with a significant conformational change in the protein on binding to DNA, with one possibility being the formation of a stable helix in the C-terminal region of R2. In addition, conformational heterogeneity identified in R2 of B-MybR2R3 bound to the tom-1-A target site may play an important role in the control of gene expression by Myb proteins.

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Preliminary investigation of the interaction between the R2R3 DNA binding domain of the oncoprotein c‐Myb and DNA fragments

Cited by 3 publications

References 0 publications

Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

Strategy to Discriminate between High and Low Affinity Bindings of Human Immunodeficiency Virus, Type 1 Integrase to Viral DNA

Solution Structure of the B-Myb DNA-Binding Domain: A Possible Role for Conformational Instability of the Protein in DNA Binding and Control of Gene Expression^,

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Preliminary investigation of the interaction between the R2R3 DNA binding domain of the oncoprotein c‐Myb and DNA fragments

Cited by 3 publications

References 0 publications

Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

Strategy to Discriminate between High and Low Affinity Bindings of Human Immunodeficiency Virus, Type 1 Integrase to Viral DNA

Solution Structure of the B-Myb DNA-Binding Domain: A Possible Role for Conformational Instability of the Protein in DNA Binding and Control of Gene Expression,

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Solution Structure of the B-Myb DNA-Binding Domain: A Possible Role for Conformational Instability of the Protein in DNA Binding and Control of Gene Expression^,