Binding of Gd³⁺ to the neuronal signalling protein calexcitin identifies an exchangeable Ca²⁺-binding site

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 R_free 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.