Structural analysis of Mg2+ and Ca2+ binding, myristoylation, and dimerization of the neuronal calcium sensor and visinin-like protein 1 (VILIP-1).

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca(2+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(2+) and Mg(2+) binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg(2+) binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K(D) = 20 μM). Unmyristoylated VILIP-1 binds two Ca(2+) sequentially at EF2 and EF3 (K(EF3) = 0.1 μM and K(EF2) = 1-4 μM), whereas myristoylated VILIP-1 binds two Ca(2+) with lower affinity (K(D) = 1.2 μM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca(2+)-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca(2+)-dependent chemical shifts and NOE patterns consistent with Ca(2+)-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca(2+) and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca(2+)-myristoyl switch to provide structural insights into Ca(2+)-induced trafficking of nicotinic acetylcholine receptors.