Abstract
Calmodulin (CaM) is a ubiquitous EF-hand calcium sensor protein that transduces calcium signals in a wide range of signaling pathways. Structural analysis of complexes with peptides has provided valuable insights into the remarkable variety in the way in which CaM interacts with and activates its targets. Among these various targets, CaM has been shown to be an essential component of a calcium-sensing regulatory apparatus for a number of voltage-gated ion channels. NMR spectroscopy has proven to be a powerful tool for the structural characterization of CaM–peptide complexes, in particular for the study of IQ motifs, which bind CaM at the basal level of calcium in cells and thereby serve to localize CaM to its sites of action. We describe here methods for the robust expression and purification of CaM isotopically enriched for NMR analysis, as well as for the complex of CaM with a peptide derived from the IQ motif sequence of the human cardiac sodium channel NaV1.5. We also describe methods for NMR analysis of titrations of CaM with IQ motif peptides to determine the stoichiometry of the complex and to identify the residues at the binding interface.
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References
Chazin WJ (2011) Relating form and function of EF-hand calcium binding proteins. Acc Chem Res 44:171–179
Bhattacharya S, Bunick CG, Chazin WJ (2004) Target selectivity in EF-hand calcium binding proteins. Biochim Biophys Acta 1742:69–79
Klee CB, Newton DL, Ni WC, Haiech J (1986) Regulation of the calcium signal by calmodulin. Ciba Found Symp 122:162–182
Chagot B, Chazin WJ (2011) Solution NMR structure of apo-calmodulin in complex with the IQ motif of human cardiac sodium channel NaV1.5. J Mol Biol 406:106–119
Fallon JL, Baker MR, Xiong L, Loy RE, Yang G, Dirksen RT, Hamilton SL, Quiocho FA (2009) Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca2+* calmodulins. Proc Natl Acad Sci U S A 106:5135–5140
Feldkamp MD, Yu L, Shea MA (2011) Structural and energetic determinants of apo calmodulin binding to the IQ motif of the NaV1.2 voltage-dependent sodium channel. Structure 19:733–747
Kim EY, Rumpf CH, Fujiwara Y, Cooley ES, Van Petegem F, Minor DL Jr (2008) Structures of CaV2 Ca2+/CaM-IQ domain complexes reveal binding modes that underlie calcium-dependent inactivation and facilitation. Structure 16:1455–1467
Mori MX, Vander Kooi CW, Leahy DJ, Yue DT (2008) Crystal structure of the CaV2 IQ domain in complex with Ca2+/calmodulin: high-resolution mechanistic implications for channel regulation by Ca2+. Structure 16:607–620
Kranz JK, Lee EK, Nairn AC, Wand AJ (2002) A direct test of the reductionist approach to structural studies of calmodulin activity: Ârelevance of peptide models of target proteins. J Biol Chem 277:16351–16354
Bahler M, Rhoads A (2002) Calmodulin signaling via the IQ motif. FEBS Lett 513:107–113
Black DJ, Leonard J, Persechini A (2006) Biphasic Ca2+-dependent switching in a calmodulin-IQ domain complex. Biochemistry 45:6987–6995
Yu FH, Catterall WA (2003) Overview of the voltage-gated sodium channel family. Genome Biol 4:207.1–207.7
Shah VN, Wingo TL, Weiss KL, Williams CK, Balser JR, Chazin WJ (2006) Calcium-dependent regulation of the voltage-gated sodium channel hH1: intrinsic and extrinsic sensors use a common molecular switch. Proc Natl Acad Sci U S A 103:3592–3597
Huth JR, Bewley CA, Jackson BM, Hinnebusch AG, Clore GM, Gronenborn AM (1997) Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR. Protein Sci 6:2359–2364
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293
Goddard TD, Kneller DG (2008) SPARKY, 3rd edn. University of California, San Francisco
Johnson BA, Blevins RA (1994) NMR View: a computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614
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Damo, S.M., Feldkamp, M.D., Chagot, B., Chazin, W.J. (2013). NMR Studies of the Interaction of Calmodulin with IQ Motif Peptides. In: Heizmann, C. (eds) Calcium-Binding Proteins and RAGE. Methods in Molecular Biology, vol 963. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-230-8_11
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DOI: https://doi.org/10.1007/978-1-62703-230-8_11
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Publisher Name: Humana Press, Totowa, NJ
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