Structural Analysis of Mg super(2+) and Ca super(2+) Binding to CaBP1, a Neuron-specific Regulator of Calcium Channels

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