title: Site-specific methionine oxidation in calmodulin affects structural integrity and interaction with Ca2+/calmodulin-dependent protein kinase II.
abstract: Methionine oxidation in the ubiquitous calcium signaling protein calmodulin (CaM) is known to disrupt downstream signaling and target CaM for proteasomal degradation. The susceptibility of CaM to oxidation in the different conformations that are sampled during calcium signaling is currently not well defined. Using an integrative mass spectrometry (MS) approach, applying both native MS and LC/MS/MS, we unravel molecular details of CaM methionine oxidation in the context of its interaction with the Ca(2+)/CaM-dependent protein kinase II (CaMKII). Sensitivity to methionine oxidation in CaM was found to vary according to the conformational state. Three methionine residues (Met71, 72, 145) show increased reactivity in calcium-saturated CaM (holo-CaM) compared to calcium-free CaM (apo-CaM), which has important consequences for oxidation-targeted proteasomal degradation. In addition, all four methionines in the C-terminal lobe (Met109, 124, 144 and 145) are found to be protected from oxidation in a peptide-based model of the CaMKII-bound conformation (cbp-CaM). We furthermore demonstrate that the oxidation of Met144 and 145 inhibits the interaction of CaM with CaMKII. cbp-CaM, in contrast to apo- and holo-CaM, maintains its ability to bind CaMKII under simulated conditions of oxidative stress and is also protected from oxidation-induced unfolding. Thus, we show that the susceptibility towards oxidation of specific residues in CaM is tightly linked to its signaling state and conformation, which has direct implications for calcium/CaM-CaMKII related signaling.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Padualaan 8, 3584 CH Utrecht, Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Snijder
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Rose
authors.forename: Reinout
authors.initials: R
authors.lastname: Raijmakers
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002853:Chromatography, Liquid; D008715:Methionine / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D053719:Tandem Mass Spectrometry
chemical_list: D002147:Calmodulin; D008715:Methionine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2