DNA-induced conformational changes are the basis for cooperative dimerization by the DNA binding domain of the retinoid X receptor

Holmbeck, S. M. A.; Dyson, H. Jane; Wright, Peter E.

doi:10.1006/jmbi.1998.2207

Cited by 65 publications

(49 citation statements)

References 28 publications

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“…Thus, in those cases where a steroid produces an FHDC, our theory predicts that GR dimerization cannot be a necessary step for gene induction and vice versa (see SI Section 7 for a theoretical justification). In support of our theory, recent NMR studies show conformational changes in DNA-bound dimers occurring after the DNA binding of monomeric receptor (18). Furthermore, experiments with both steroid receptors (19)(20)(21)(22) and helix-loop-helix zipper transcription factors (23, 24) strongly suggest that the majority of receptor binding to HREs occurs not by preformed dimers but by monomers that then form dimers on the DNA.…”

Section: Positionsupporting

confidence: 80%

A theoretical framework for gene induction and experimental comparisons

Ong

Blackford

Kagan

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

113

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Ligand-mediated gene induction by steroid receptors is a multistep process characterized by a dose-response curve for gene product that follows a first-order Hill equation. This behavior has classically been explained by steroid binding to receptor being the rate-limiting step. However, this predicts a constant potency of gene induction (EC 50 ) for a given receptor-steroid complex, which is challenged by the findings that various cofactors/reagents can alter this parameter in a gene-specific manner. These properties put strong constraints on the mechanisms of gene induction and raise two questions: How can a first-order Hill dose-response curve (FHDC) arise from a multistep reaction sequence, and how do cofactors modify potency? Here we introduce a theoretical framework in which a sequence of steps yields an FHDC for the final product as a function of the initial agonist concentration. An exact determination of all constants is not required to describe the final FHDC. The theory predicts mechanisms for cofactor/reagent effects on gene-induction potency and maximal activity and it assigns a relative order to cofactors in the sequence of steps. The theory is supported by several observations from glucocorticoid receptor-mediated gene induction. It identifies the mechanism and matches the measured dose-response curves for different concentrations of the combination of cofactor Ubc9 and receptor. It also predicts that an FHDC cannot involve the DNA binding of preformed receptor dimers, which is validated experimentally. The theory is general and can be applied to any biochemical reaction that shows an FHDC.dose-response | Michaelis-Menten | gene expression | steroid receptors | glucocorticoids | pharmacology I n ligand-mediated gene induction, the amount of gene expressed depends on the amount of ligand present. Thus, the specific shape and properties of the dose-response curve of gene induction, which is of crucial importance for development, differentiation, and homeostasis in many biological systems, provide a quantitative means for probing the gene-induction process. In many cases, the dose-response curve in gene induction obeys a sigmoidal curve, but not all sigmoidal curves have the same shape. For example, a dose-response curve obeying a first-order Hill equation or function (Hill coefficient equal to 1) goes from 10 to 90% of maximum activity over an 81-fold change in ligand concentration, whereas only a 9-fold change is required in a secondorder Hill function, which thus has a different shape (Fig. S1). (A first-order Hill function is sometimes called a Michaelis-Menten function.) Depending upon the shape of the dose-response curve, the responsiveness of gene induction to the same variation in ligand concentration will differ greatly. In addition to the shape, the position or potency [i.e., concentration required for 50% of maximal response (EC 50 )] and maximum activity (A max ) of the dose-response curve are required to specify the amount of gene expressed for a given amount of ligand. Despite the vital...

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Section: Positionsupporting

confidence: 80%

A theoretical framework for gene induction and experimental comparisons

Ong

Blackford

Kagan

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

113

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“…These assignments allowed chemical shift changes upon DNA binding to be mapped to specific regions of the protein backbone. Similar chemical shift mapping strategies have been widely used to localise intermolecular binding surfaces in other DNA/protein systems (31)(32)(33). In the present case, binding of Ada-C to either ss-or dsDNA involved a similar pattern of backbone amide chemical shift changes.…”

Section: Resultsmentioning

confidence: 74%

DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase

Verdemato¹

2000

Nucleic Acids Research

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The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O 6 -alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O 6 -methylguanine (O 6 meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.

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“…NIH-PA Author Manuscript NIH-PA Author Manuscript that they may be important characteristics of genetic regulation (e.g., (11,36,37,(59)(60)(61)(62)(63)(64)(65)). In LacI and its homologues, the hinge helix is responsible for both of these features.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

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A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short α-helices when bound to DNA, but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge helix folding and/or hinge•hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality. KeywordsLacI; helical propensity; allostery; DNA binding Protein folding has long been a fundamental issue in biological sciences. Beyond the general question of how a polypeptide sequence adopts a three-dimensional structure, coupled folding and binding have been identified as a key feature of partially unstructured proteins in multiple regulatory processes (e.g., transcription regulation, signal transduction, membrane transport and signaling (6-9)). Indeed, more and more intrinsically disordered proteins are found to fold upon binding to their target partners, ligands, or even small ions (10-13). In the case of transcription regulation, mutual cooperative folding of both protein and DNA has been † This work was supported by NIH Grant GM22441 and Robert A. Welch Grant C-576 to Kathleen Shive Matthews. Liskin Swint-Kruse is supported by NIH Grant P20 RR17708 from the Institutional Development Award program of the National Center for Research Resources.*To whom correspondence should be addressed. Telephone: 713−348−4871; Fax: 713−348−6149; Email: E-mail: ksm@bioc.rice.edu.. observed (6,11,14,15). In this paper, coupled folding is explored in greater detail for the hinge helix of the lactose repressor protein (LacI) 1 . NIH Public AccessLacI is a premier model for the regulation of gene expression and allosteric transition in biological systems (2) 2 . This 150-kDa homotetramer is a dimer of functionally independent dimers, with ...

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DNA-induced conformational changes are the basis for cooperative dimerization by the DNA binding domain of the retinoid X receptor

Cited by 65 publications

References 28 publications

A theoretical framework for gene induction and experimental comparisons

A theoretical framework for gene induction and experimental comparisons

DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

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