HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 18, 18008-18014, May 6, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/18/18008    most recent M500338200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Osawa, M. Articles by Ames, J. B. Search for Related Content PubMed PubMed Citation Articles by Osawa, M. Articles by Ames, J. B. Social Bookmarking                      What's this?

Mg²⁺ and Ca²⁺ Differentially Regulate DNA Binding and Dimerization of DREAM^*

Masanori Osawa, Alexandra Dace, Kit I. Tong¶, Aswani Valiveti, Mitsuhiko Ikura¶, and James B. Ames||

From the Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850 and the ^¶Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Ontario M5G 2M9, Canada

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca²⁺ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca²⁺ binding to the various mutants revealed that, in the absence of Mg²⁺, Ca²⁺ binds independently and sequentially to EF-3 (H = -2.4 kcal/mol), EF-4 (H = +5.2 kcal/mol), and EF-2 (H = +1 kcal/mol). By contrast, only two Ca²⁺ bind to DREAM in the presence of physiological levels of Mg²⁺ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg²⁺. ITC measurements demonstrate that one Mg²⁺ binds enthalpically with high affinity (K_d = 13 µM and H = -0.79 kcal/mol) and two or more Mg²⁺ bind entropically in the millimolar range. Size-exclusion chromatography studies revealed that Mg²⁺ stabilizes DREAM as a monomer, whereas Ca²⁺ induces protein dimerization. Electrophoretic mobility shift assays indicated that Mg²⁺ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca²⁺ levels, suggesting that binding of a single Ca²⁺ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca²⁺ and Mg²⁺ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg²⁺ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca²⁺-induced protein dimerization disrupts DNA binding.

Received for publication, January 11, 2005 , and in revised form, February 22, 2005.

^* This work was supported in part by a Beckman Foundation Young Investigator Award (to J. B. A.) and Grants NS45909 and EY12347 from the National Institutes of Health (to J. B. A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by postdoctoral fellowships from the Human Frontier Science Program and Uehara Memorial Foundation.

^|| To whom correspondence should be addressed: The Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Dr., Rockville, MD 20850. Tel.: 301-738-6120; Fax: 301-738-6255; E-mail: james{at}carb.nist.gov.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. Li, J. Chan, F. Haeseleer, K. Mikoshiba, K. Palczewski, M. Ikura, and J. B. Ames Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1 J. Biol. Chem., January 23, 2009; 284(4): 2472 - 2481. [Abstract] [Full Text] [PDF]

				M. C. Kim, W. S. Chung, D.-J. Yun, and M. J. Cho Calcium and Calmodulin-Mediated Regulation of Gene Expression in Plants Mol Plant, January 6, 2009; (2009) ssn091v1. [Abstract] [Full Text] [PDF]

				J. Schwenk, G. Zolles, N. G. Kandias, I. Neubauer, H. Kalbacher, M. Covarrubias, B. Fakler, and D. Bentrop NMR Analysis of KChIP4a Reveals Structural Basis for Control of Surface Expression of Kv4 Channel Complexes J. Biol. Chem., July 4, 2008; 283(27): 18937 - 18946. [Abstract] [Full Text] [PDF]

				R. Kushwaha, A. Singh, and S. Chattopadhyay Calmodulin7 Plays an Important Role as Transcriptional Regulator in Arabidopsis Seedling Development PLANT CELL, July 1, 2008; 20(7): 1747 - 1759. [Abstract] [Full Text] [PDF]

				B. Cebolla, A. Fernandez-Perez, G. Perea, A. Araque, and M. Vallejo DREAM Mediates cAMP-Dependent, Ca2+-Induced Stimulation of GFAP Gene Expression and Regulates Cortical Astrogliogenesis J. Neurosci., June 25, 2008; 28(26): 6703 - 6713. [Abstract] [Full Text] [PDF]

				Y. Tozawa, A. Nozawa, T. Kanno, T. Narisawa, S. Masuda, K. Kasai, and H. Nanamiya Calcium-activated (p)ppGpp Synthetase in Chloroplasts of Land Plants J. Biol. Chem., December 7, 2007; 282(49): 35536 - 35545. [Abstract] [Full Text] [PDF]

				K. T. Buchanan, J. B. Ames, S. H. Asfaw, J. N. Wingard, C. L. Olson, P. T. Campana, A. P. U. Araujo, and D. M. Engman A Flagellum-specific Calcium Sensor J. Biol. Chem., December 2, 2005; 280(48): 40104 - 40111. [Abstract] [Full Text] [PDF]

				J. N. Wingard, J. Chan, I. Bosanac, F. Haeseleer, K. Palczewski, M. Ikura, and J. B. Ames Structural Analysis of Mg2+ and Ca2+ Binding to CaBP1, a Neuron-specific Regulator of Calcium Channels J. Biol. Chem., November 11, 2005; 280(45): 37461 - 37470. [Abstract] [Full Text] [PDF]