title: X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure-function studies of a neuronal calcium-signalling protein.
abstract: The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, England.
authors.forename: P T
authors.initials: PT
authors.lastname: Erskine
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, England.
authors.forename: A
authors.initials: A
authors.lastname: Fokas
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, England.
authors.forename: C
authors.initials: C
authors.lastname: Muriithi
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, England.
authors.forename: H
authors.initials: H
authors.lastname: Rehman
authors.affiliation: Centre of Biological Sciences, University of Southampton, Southampton SO17 1BJ, England.
authors.forename: L A
authors.initials: LA
authors.lastname: Yates
authors.affiliation: Centre of Biological Sciences, University of Southampton, Southampton SO17 1BJ, England.
authors.forename: A
authors.initials: A
authors.lastname: Bowyer
authors.affiliation: Centre of Biological Sciences, University of Southampton, Southampton SO17 1BJ, England.
authors.forename: I S
authors.initials: IS
authors.lastname: Findlow
authors.affiliation: Centre of Biological Sciences, University of Southampton, Southampton SO17 1BJ, England.
authors.forename: R
authors.initials: R
authors.lastname: Hagan
authors.affiliation: Centre of Biological Sciences, University of Southampton, Southampton SO17 1BJ, England.
authors.forename: J M
authors.initials: JM
authors.lastname: Werner
authors.affiliation: Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, England.
authors.forename: A J
authors.initials: AJ
authors.lastname: Miles
authors.affiliation: Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, England.
authors.forename: B A
authors.initials: BA
authors.lastname: Wallace
authors.affiliation: Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, England.
authors.forename: S A
authors.initials: SA
authors.lastname: Wells
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, England.
authors.forename: S P
authors.initials: SP
authors.lastname: Wood
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, England.
authors.forename: J B
authors.initials: JB
authors.lastname: Cooper
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D049832:Decapodiformes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D002135:Calcium-Binding Proteins; D009419:Nerve Tissue Proteins
keywords: EF-hand; calcium signalling; learning; memory; neuronal plasticity