Department of Chemistry, University of California, Davis, California 95616, USA.
J Biol Chem. 2011 Feb 25;286(8):6354-66. doi: 10.1074/jbc.M110.173724. Epub 2010 Dec 17.
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.
视蛋白样蛋白 1（VILIP-1）属于神经元钙传感器家族的 Ca(2+)-豆蔻酰开关蛋白，可调节大脑和视网膜中的信号转导。在此，我们分析了 Ca(2+)和 Mg(2+)结合、表征了金属诱导的构象变化，并确定了豆蔻酰化和二聚化的结构效应。EF3 处的 Mg(2+)（ΔH = +1.8 kcal/mol 和 K(D) = 20 μM）与 VILIP-1 功能结合。未豆蔻酰化的 VILIP-1 先后在 EF2 和 EF3 处结合两个 Ca(2+)（K(EF3) = 0.1 μM 和 K(EF2) = 1-4 μM），而豆蔻酰化的 VILIP-1 以较低亲和力（K(D) = 1.2 μM）和正协同性（Hill 斜率 = 1.5）结合两个 Ca(2+)。NMR 分配和结构分析表明，Ca(2+) 自由的 VILIP-1 包含一个像 recoverin 那样的被隔离的豆蔻酰基。连接的豆蔻酸盐的 NMR 共振表现出 Ca(2+)依赖性化学位移和 NOE 模式，与 Ca(2+)诱导的豆蔻酸挤出一致。VILIP-1 在溶液中独立于 Ca(2+)和豆蔻酰化形成二聚体。二聚化位点由 EF4 中的残基和 EF3 与 EF4 之间的环区组成，这一点通过突变得到了证实。我们展示了 VILIP-1 二聚体和 Ca(2+)-豆蔻酰开关的结构，为 Ca(2+)诱导的烟碱型乙酰胆碱受体运输提供了结构见解。
