title: Binding of Gd(3+) to the neuronal signalling protein calexcitin identifies an exchangeable Ca(2+)-binding site.
abstract: Calexcitin was first identified in the marine snail Hermissenda crassicornis as a neuronal-specific protein that becomes upregulated and phosphorylated in associative learning. Calexcitin possesses four EF-hand motifs, but only the first three (EF-1 to EF-3) are involved in binding metal ions. Past work has indicated that under physiological conditions EF-1 and EF-2 bind Mg(2+) and Ca(2+), while EF-3 is likely to bind only Ca(2+). The fourth EF-hand is nonfunctional owing to a lack of key metal-binding residues. The aim of this study was to use a crystallographic approach to determine which of the three metal-binding sites of calexcitin is most readily replaced by exogenous metal ions, potentially shedding light on which of the EF-hands play a `sensory' role in neuronal calcium signalling. By co-crystallizing recombinant calexcitin with equimolar Gd(3+) in the presence of trace Ca(2+), EF-1 was shown to become fully occupied by Gd(3+) ions, while the other two sites remain fully occupied by Ca(2+). The structure of the Gd(3+)-calexcitin complex has been refined to an R factor of 21.5% and an Rfree of 30.4% at 2.2 Å resolution. These findings suggest that EF-1 of calexcitin is the Ca(2+)-binding site with the lowest selectivity for Ca(2+), and the implications of this finding for calcium sensing in neuronal signalling pathways are discussed.
authors.affiliation: Division of Medicine, UCL, Gower Street, London WC1E 6BT, England.
authors.forename: Lucas
authors.initials: L
authors.lastname: Chataigner
authors.affiliation: Division of Medicine, UCL, Gower Street, London WC1E 6BT, England.
authors.forename: Jingxu
authors.initials: J
authors.lastname: Guo
authors.affiliation: Division of Medicine, UCL, Gower Street, London WC1E 6BT, England.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Erskine
authors.affiliation: Division of Medicine, UCL, Gower Street, London WC1E 6BT, England.
authors.forename: Alun R
authors.initials: AR
authors.lastname: Coker
authors.affiliation: Division of Medicine, UCL, Gower Street, London WC1E 6BT, England.
authors.forename: Steve P
authors.initials: SP
authors.lastname: Wood
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Gombos
authors.affiliation: Division of Medicine, UCL, Gower Street, London WC1E 6BT, England.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cooper
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D005682:Gadolinium / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D002135:Calcium-Binding Proteins; D005682:Gadolinium; D002118:Calcium
keywords: EF-hand; co-crystallization; heavy-atom complex; neuronal calcium signalling; protein structure