Fast methioninebased solution structure determina

Authors: Jessica L Gifford Hiroaki Ishida Hans J Vogel

Publish Date: 2011/03/01

Volume: 50, Issue: 1, Pages: 71-81

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Abstract

Here we present a novel NMR method for the structure determination of calciumcalmodulin Ca2+CaMpeptide complexes from a limited set of experimental restraints A comparison of solved CaMpeptide structures reveals invariability in CaM’s backbone conformation and a structural plasticity in CaM’s domain orientation enabled by a flexible linker Knowing this the collection and analysis of an extensive set of NOESY spectra is redundant Although RDCs can define CaM domain orientation in the complex they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs Here we employ a specific isotope labeling strategy in which the role of methionine in CaMpeptide interactions is exploited to collect these critical NOEs By 1H 13Clabeling the methyl groups of deuterated methionine against a 2H 12C background we can acquire a 13Cedited NOESY characterized by simplified easily analyzable spectra Together with measured CaM backbone HNN RDCs and intrapeptide NOEbased distances these intermolecular NOEs provide restraints for a low temperature torsionangle dynamics and simulated annealing protocol used to calculate the complex structure We have applied our method to a CaM complex previously solved through Xray crystallography Ca2+CaM bound to the CaM kinase I peptide PDB code 1MXE The resulting structure has a backbone RMSD of 16 Å to that previously published We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome In addition to having application for fast complex structure determination this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully 13C 15Nlabeled CaM failsThe authors wish to thank Deane D McIntyre for the synthesis of 1Hαε 13Cε 2Hmethionine as well as for spectrometer maintenance Renee Otten for helpful discussions and the Canadian Institutes of Health Research for operating support JLG is the recipient of postgraduate scholarships from the National Science and Engineering Research Council and Alberta Ingenuity Fund as well as a Killam Fellowship HJV is an Alberta Heritage Foundation for Medical Research Scientist

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