title: 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 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.
authors.affiliation: Department of Chemistry, University of California, Davis, California 95616, USA.
authors.forename: Congmin
authors.initials: C
authors.lastname: Li
authors.forename: Wensheng
authors.initials: W
authors.lastname: Pan
authors.forename: Karl H
authors.initials: KH
authors.lastname: Braunewell
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
mesh_terms: D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D054878:Lipoylation / Q000502:physiology*; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D019814:Myristic Acid / Q000737:chemistry* / Q000378:metabolism; D051596:Neurocalcin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000502:physiology; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D051596:Neurocalcin; D011978:Receptors, Nicotinic; C496688:VSNL1 protein, human; D019814:Myristic Acid; D008274:Magnesium; D002118:Calcium