نبذة مختصرة : 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 Mg2+ and Ca2+, while EF-3 is likely to bind only Ca2+. 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 Gd3+ in the presence of trace Ca2+, EF-1 was shown to become fully occupied by Gd3+ ions, while the other two sites remain fully occupied by Ca2+. The structure of the Gd3+-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 Ca2+-binding site with the lowest selectivity for Ca2+, and the implications of this finding for calcium sensing in neuronal signalling pathways are discussed.
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