Binding of Gd³⁺to the neuronal signalling protein calexcitin identifies an exchangeable Ca²⁺-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 R free 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.

The X-ray structure of juvenile hormone diol kinase from the silkworm Bombyx mori

“…However, a run of the MrBUMP molecular-replacement pipeline (Keegan et al, 2018), which called upon ECOD (Cheng, Schaeffer et al, 2014) to identify any possible domain-based search models, picked out two clear domains within the related homologues. These were used as search models for Phaser (McCoy et al, 2005) within MrBUMP, which yielded a solution based on domains from PDB entries 5f6t and 4ndd (Chataigner et al, 2016;Erskine et al, 2015), both of which are structures of L. pealei calexcitin. In spite of the presence of six copies of the protein molecule in the asymmetric unit (Kantardjieff & Rupp, 2003), Phaser was able to slowly accumulate the solution.…”