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J. Biol. Chem., Vol. 280, Issue 45, 37461-37470, November 11, 2005

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Structural Analysis of Mg²⁺ and Ca²⁺ Binding to CaBP1, a Neuron-specific Regulator of Calcium Channels^*

Jennifer N. Wingard, Jenny Chan, Ivan Bosanac, Françoise Haeseleer¶, Krzysztof Palczewski¶||**1, Mitsuhiko Ikura2, and James B. Ames3

From the Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, Division of Signaling Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada, and the Departments of ^¶Ophthalmology, ^||Pharmacology, and ^**Chemistry, University of Washington, Seattle, Washington 98195

CaBP1 (calcium-binding protein 1) is a 19.4-kDa protein of the EF-hand superfamily that modulates the activity of Ca²⁺ channels in the brain and retina. Here we present data from NMR, microcalorimetry, and other biophysical studies that characterize Ca²⁺ binding, Mg²⁺ binding, and structural properties of recombinant CaBP1 purified from Escherichia coli. Mg²⁺ binds constitutively to CaBP1 at EF-1 with an apparent dissociation constant (K_d) of 300 µM. Mg²⁺ binding to CaBP1 is enthalpic (H = –3.725 kcal/mol) and promotes NMR spectral changes, indicative of a concerted Mg²⁺-induced conformational change. Ca²⁺ binding to CaBP1 induces NMR spectral changes assigned to residues in EF-3 and EF-4, indicating localized Ca²⁺-induced conformational changes at these sites. Ca²⁺ binds cooperatively to CaBP1 at EF-3 and EF-4 with an apparent K_d of 2.5 µM and a Hill coefficient of 1.3. Ca²⁺ binds to EF-1 with low affinity (K_d >100 µM), and no Ca²⁺ binding was detected at EF-2. In the absence of Mg²⁺ and Ca²⁺, CaBP1 forms a flexible molten globule-like structure. Mg²⁺ and Ca²⁺ induce distinct conformational changes resulting in protein dimerization and markedly increased folding stability. The unfolding temperatures are 53, 74, and 76 °C for apo-, Mg²⁺-bound, and Ca²⁺-bound CaBP1, respectively. Together, our results suggest that CaBP1 switches between structurally distinct Mg²⁺-bound and Ca²⁺-bound states in response to Ca²⁺ signaling. Both conformational states may serve to modulate the activity of Ca²⁺ channel targets.

Received for publication, August 3, 2005

^* This work was supported in part by Grants EY012347 and NS045909 (to J. B. A.) and EY08061 (to K. P.) from the National Institutes of Health. 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.

¹ Present address: Dept. of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4965.

² Supported by Canadian Institutes of Health Research and holds Canada Research Chair in Cancer Structural Biology.

³ To whom correspondence should be addressed: Center for Advanced Research in Biotechnology, Rockville, MD 20850. Tel.: 240-314-6120; Fax: 240-314-6255; E-mail: james{at}carb.nist.gov.

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