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

Abstract

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ and Mg2+ binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg2+ binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K D = 20 μM). Unmyristoylated VILIP-1 binds two Ca2+ sequentially at EF2 and EF3 (KEF3 = 0.1 μM and KEF2 = 1-4 μM), whereas myristoylated VILIP-1 binds two Ca2+ with lower affinity (KD = 1.2 μM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca2+-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca2+-dependent chemical shifts and NOE patterns consistent with Ca2+-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca2+ 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 Ca2+-myristoyl switch to provide structural insights into Ca2+-induced trafficking of nicotinic acetylcholine receptors. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.