X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure–function studies of a neuronal calcium-signalling protein

PT Erskine, A Fokas, C Muriithi, H Rehman, LA Yates, A Bowyer, IS Findlow, R Hagan, JM Miles, BA Wallace, Stephen Wells, SP Wood, JB Cooper

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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 calciumdependent
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
Original languageEnglish
Pages (from-to)615-631
JournalActa Crystallographica Section D-Biological Crystallography
Issue number3
Publication statusPublished - Apr 2015


  • calcium signalling; neuronal plasticity; learning; memory; EF-hand
  • protein flexibility
  • protein rigidity


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