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University of Bath Citation for published version: Erskine, PT, Fokas, A, Muriithi, C, Rehman, H, Yates, LA, Bowyer, A, Findlow, IS, Hagan, R, Miles, JM, Wallace, BA, Wells, S, Wood, SP & Cooper, JB 2015, 'X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure–function studies of a neuronal calcium-signalling protein', Acta Crystallographica Section D-Biological Crystallography, vol. 71, no. 3, pp. 615-631. https://doi.org/10.1107/S1399004714026704 DOI: 10.1107/S1399004714026704 Publication date: 2015 Document Version Publisher's PDF, also known as Version of record Link to publication University of Bath General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 13. May. 2019 electronic reprint Acta Crystallographica Section D Biological Crystallography ISSN 1399-0047 X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure–function studies of a neuronal calcium-signalling protein P. T. Erskine, A. Fokas, C. Muriithi, H. Rehman, L. A. Yates, A. Bowyer, I. S. Findlow, R. Hagan, J. M. Werner, A. J. Miles, B. A. Wallace, S. A. Wells, S. P. Wood and J. B. Cooper Acta Cryst. (2015). D71, 615–631 Copyright c International Union of Crystallography Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr. For further information see http://journals.iucr.org/services/authorrights.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the struc- tures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure–function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallo- graphic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crys- tallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability. Crystallography Journals Online is available from journals.iucr.org Acta Cryst. (2015). D71, 615–631 Erskine et al. · Calexcitin research papers X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure–function studies of a neuronal calcium-signalling protein ISSN 1399-0047 P. T. Erskine,a A. Fokas,a C. Muriithi,a H. Rehman,a L. A. Yates,b‡ A. Bowyer,b I. S. Findlow,b R. Hagan,b J. M. Werner,b A. J. Miles,c B. A. Wallace,c S. A. Wells,d S. P. Wooda and J. B. Coopera* Received 16 November 2014 Accepted 4 December 2014 aLaboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, England, bCentre of Biological Sciences, University of Southampton, Southampton SO17 1BJ, England, cInstitute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, England, and dDepartment of Chemistry, University of Bath, Claverton Down, ‡ Present address: Division of Structural Bath BA2 7AY, England. *Correspondence e-mail: [email protected] Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, England. The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated Keywords: calcium signalling; neuronal following a Pavlovian conditioning protocol. Subsequent studies showed that plasticity; learning; memory; EF-hand. calexcitin regulates the voltage-dependent potassium channel and the calcium- dependent potassium channel as well as causing the release of calcium ions from PDB references: calexcitin, T188D mutant, 4ndc; T61D+T188D double mutant, 4ndd; the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal T61D mutant, 4ndb 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 Supporting information: this article has domains of which are known to separate upon calcium binding, allowing supporting information at journals.iucr.org/d 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. 1. Introduction The protein calexcitin was originally identified in the photo- receptor neurons of the marine snail Hermissenda crassicornis as a 20 kDa cytosolic protein that became up-regulated and phosphorylated following a Pavlovian training protocol # 2015 International Union of Crystallography (Nelson et al., 1990). Hermissenda are normally attracted Acta Cryst. (2015). D71, 615–631 doi:10.1107/S1399004714026704 615 electronic reprint research papers towards light but are repelled by movement, which causes foot to bind to the protein with submicromolar affinity and they retraction. The snails become conditioned to retract upon induce significant conformational changes within the physio- exposure to light (normally an attractive stimulus) if they are logical Ca2+ concentration range. This has been demonstrated simultaneously exposed to both light and movement (e.g. on by circular dichroism (CD), fluorescence, NMR and infrared an illuminated turntable). The up-regulation of calexcitin was spectroscopy (Gombos et al., 2001; Ascoli et al., 1997). correlated with exposure to both light and movement and Calexcitin shares significant sequence identity (almost 40%) not with each separate stimulus alone. Electrophysiological with juvenile hormone diol kinase (JHDK), an insect enzyme studies involving micro-injection of the purified calexcitin that uses ATP or GTP to phosphorylate its polyisoprene protein into Hermissenda photoreceptors showed that it substrate (Maxwell, Welsh, Horodyski et al., 2002; Maxwell, regulated the voltage-dependent potassium channel and the Welch & Schooley, 2002; Li et al., 2005). Intriguingly, the calcium-dependent potassium channel and was capable of activity of JHDK is inhibited by the presence of calcium, reproducing the electrophysiological effects of learning in suggesting that the calcium-free or partly decalcified form of neurons. The protein was found to bind calcium and there is the protein may be the catalytically active species. evidence that it has GTPase activity (Alkon & Nelson, 1990; The three-dimensional structure of L. pealei calexcitin has Nelson et al., 1994). been determined by X-ray analysis at a resolution of 1.8 A˚ Calexcitin was first isolated from the optic lobe of the long- (Beaven et al., 2005; Erskine et al., 2006). The overall structure finned squid Loligo pealei (Nelson et al., 1996). It has been is predominantly helical and compact, with a pronounced shown that the protein kinase C (PKC)-dependent phos- hydrophobic core between the N- and C-terminal domains of phorylation of calexcitin causes it to translocate to the cell the molecule. The structure consists of four EF-hand motifs membrane, where its effects on membrane excitability
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