X-ray, Spectroscopy and Normal-mode Dynamics of Calexcitin:
Structure-function Studies of a Neuronal Calcium-signalling Protein

This is a CCP5-funded collaborative work that allows Dr Stephen Wells of University of Bath to apply rapid modelling methods, combining with static crystal structure data and NMR data to carry out structure determination, analysis and publication.

The first paper on this work has been accepted for publication [1] reporting both new protein crystal structures (mutants of calexcitin) and analysis of their geometry rigidity and flexibility using the rapid FIRST/FRODA+Elnemo approach.

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. Our crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, whose N- and C-terminal domains 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 (Thr 61 and Thr 188).

We have introduced Thr-to-Asp mutations which mimic phosphorylation of the protein and have determined crystal structures of the corresponding single and double mutants which suggest that the C-terminal phosphorylation site (Thr 188) exerts the greatest effects on the protein structure. We have also conducted extensive NMR studies which demonstrate that in solution the wild-type protein predominantly adopts a more open conformation than the crystallographic studies have indicated and, accordingly, our 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 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 weakest of which is most likely 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 calexcitin�s 3 calcium-binding sites 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.

  

Figures above illustrates the modelling of the intrinsic flexibility of wild-type and mutant forms of calexcitin: Normal mode dynamics in wild-type (left) and T188D mutant (right) calexcitin structures. In each case the ribbon bundle shows the main chain geometry of conformers generated by geometric simulations which are biased parallel and antiparallel to the ten lowest frequency nontrivial normal modes (7 to 16) until the amplitude is limited by steric or non-covalent interaction constraints. The chain is �rainbowed� from blue (N-terminus) to red (C-terminus). Simulations include residues 6-191; i.e. residues 1 - 5 were omitted as otherwise multiple modes localize on the N-terminal tail. The T188D structure displays a slightly greater range of flexible motion, particularly around the C-terminal region (orange, red) and the nearby N-terminal helix (dark blue).

[1] Peter Erskine, Alexander Fokas, Caroline Muriithi, Hannah Rehman, Luke Yates, Alexandra Bowyer, Stuart Findlow, Robert Hagan, JoernWerner, Andrew Miles, Stephen Wells, SteveWood and Jon Cooper*
Acta Crystallographica D. To be published.

 

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