Spectroscopic Study of the Conformational Changes Involved in the Activation of CaMKII

Abstract

The CaMKII enzyme is involved in diverse biological processes including control of heart rate and synaptic plasticity. CaMKII is activated by binding of calmodulin (CaM) and ATP. Its intricate structure and dynamics are thought to underlie its mode of self-regulation. The enzyme consists of catalytic, regulatory (auto-inhibitory), and association domains. Unregulated CaMKII activity has been implicated in cardiac pathologies. As such, a better understanding of the activation mechanism of the enzyme could contribute to the development of CaMKII-specific inhibitors. The hypothesis tested was that binding of ATP to CaMKII alters the flexibility of the regulatory domain and induces conformational rearrangements in the catalytic domain of the kinase. Toward this end, studies were made on monomeric constructs lacking the association domain. Spin labels were placed at specific positions in the regulatory and catalytic domains for distance measurements by Double Electron-Electron Resonance (DEER) in basal, +ATP, and +CaM conditions. Additionally, the effect of ATP on the CaM-binding region was studied on a per-residue basis by NMR. The findings demonstrate 1) a re-structuring of the regulatory domain upon CaM binding, 2) a change in regulatory domain dynamics due to ATP binding, consistent with increased exposure of the CaM-binding segment to the solvent, and 3) shorter distances within the catalytic domain after addition of ATP, in concordance with a closed catalytic domain conformation.