title: Peptide binding by a fragment of calmodulin composed of EF-hands 2 and 3.
abstract: Calmodulin (CaM) is composed of two EF-hand domains tethered by a flexible linker. Upon Ca2+-binding, a fragment of CaM encompassing EF-hands 2 and 3 (CaM2/3; residues 46-113) folds into a structure remarkably similar to the N- and C-domains of CaM. In this study, we demonstrate that Ca2+-ligated CaM2/3 can also bind to a peptide representing the CaM-recognition sequence of skeletal muscle myosin light chain kinase (M13) with an equimolar stoichiometry and a dissociation constant of 0.40 +/- 0.05 microM. On the basis of an analytical ultracentrifugation measurement, the resulting complex exists as an equilibrium mixture of 2:2 heterotetrameric and 1:1 heterodimeric species. Chemical shift perturbation mapping indicates that, similar to CaM, the peptide associates with a hydrophobic groove crossing both EF-hands in CaM2/3. However, upon binding the M13 peptide, many residues in CaM2/3 yielded two equal intensity NMR signals with the same 15N relaxation properties. Thus, the 2:2 CaM2/3-M13 tetramer, which predominates under the conditions used for these studies, is asymmetric with each component adopting spectroscopically distinguishable conformations within the complex. CaM2/3 also weakly stimulates the phosphatase activity of calcineurin and inhibits stimulation by native CaM. These studies highlight the remarkable plasticity of EF-hand association and expand the diverse repertoire of mechanisms possible for CaM-target protein interactions.
authors.affiliation: Faculty of Pharmaceutical Sciences, Division of Biomolecular and Pharmaceutical Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Ted M
authors.initials: TM
authors.lastname: Lakowski
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Lee
authors.forename: Barbara
authors.initials: B
authors.lastname: Lelj-Garolla
authors.forename: Mark
authors.initials: M
authors.lastname: Okon
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Reid
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
mesh_terms: D001665:Binding Sites; D019703:Calcineurin / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020832:EF Hand Motifs; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009219:Myosin-Light-Chain Kinase / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary
chemical_list: D002147:Calmodulin; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D019703:Calcineurin