Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca(2+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(2+) binding, characterize Ca(2+)-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca(2+) bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (K(D) = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca(2+)-bound VILIP-3, whereas Ca(2+)-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca(2+)-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C(14) methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.