title: Structural analysis of Mg2+ and Ca2+ binding to CaBP1, a neuron-specific regulator of calcium channels.
abstract: CaBP1 (calcium-binding protein 1) is a 19.4-kDa protein of the EF-hand superfamily that modulates the activity of Ca(2+) channels in the brain and retina. Here we present data from NMR, microcalorimetry, and other biophysical studies that characterize Ca(2+) binding, Mg(2+) binding, and structural properties of recombinant CaBP1 purified from Escherichia coli. Mg(2+) binds constitutively to CaBP1 at EF-1 with an apparent dissociation constant (K(d)) of 300 microm. Mg(2+) binding to CaBP1 is enthalpic (DeltaH = -3.725 kcal/mol) and promotes NMR spectral changes, indicative of a concerted Mg(2+)-induced conformational change. Ca(2+) binding to CaBP1 induces NMR spectral changes assigned to residues in EF-3 and EF-4, indicating localized Ca(2+)-induced conformational changes at these sites. Ca(2+) binds cooperatively to CaBP1 at EF-3 and EF-4 with an apparent K(d) of 2.5 microM and a Hill coefficient of 1.3. Ca(2+) binds to EF-1 with low affinity (K(d) >100 microM), and no Ca(2+) binding was detected at EF-2. In the absence of Mg(2+) and Ca(2+), CaBP1 forms a flexible molten globule-like structure. Mg(2+) and Ca(2+) induce distinct conformational changes resulting in protein dimerization and markedly increased folding stability. The unfolding temperatures are 53, 74, and 76 degrees C for apo-, Mg(2+)-bound, and Ca(2+)-bound CaBP1, respectively. Together, our results suggest that CaBP1 switches between structurally distinct Mg(2+)-bound and Ca(2+)-bound states in response to Ca(2+) signaling. Both conformational states may serve to modulate the activity of Ca(2+) channel targets.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, 20850, USA.
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Wingard
authors.forename: Jenny
authors.initials: J
authors.lastname: Chan
authors.forename: Ivan
authors.initials: I
authors.lastname: Bosanac
authors.forename: Françoise
authors.initials: F
authors.lastname: Haeseleer
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D019281:Dimerization; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001059:Apoproteins; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; C085577:Ca2+-binding protein-1; D008274:Magnesium; D002118:Calcium