A domain-swapped CaMKII conformation facilitates linker-mediated allosteric regulation

Memory formation, fertilization, and cardiac function rely on precise Ca²⁺ signaling and subsequent Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation. Ca²⁺ sensitivity of the four CaMKII paralogs in mammals has been linked to the length of the variable linker region that is a hot spot for alternative splicing. In this study, we determined that the position of charged residues within the linker modulates the Ca²⁺/CaM sensitivity. We present an X-ray crystal structure of the full-length CaMKIIδ holoenzyme consisting of domain-swapped dimers within a dodecameric complex. In this structure, the kinase domain of one subunit is docked onto the hub domain of an adjacent subunit, providing an additional interface within the holoenzyme. Mutations at the hub equatorial and lateral interfaces led to alterations in the stoichiometry of CaMKII holoenzyme as well as Ca²⁺/CaM sensitivity. Using molecular dynamics (MD) to compare domain-swapped to non-domain-swapped CaMKIIs, we demonstrate that the domain-swapped conformation facilitates an interaction between the calmodulin binding region and the linker region. Based on MD simulations and small-angle X-ray scattering (SAXS) measurements, we propose a model where the position of charges on the linker region drives an interaction with the regulatory segment that modulates the degree of autoinhibition. Finally, we use live-cell imaging to show that the activation profiles we observe in vitro are recapitulated in cells. Our findings provide a new framework for understanding allosteric regulation of CaMKII by the linker region in Ca²⁺-sensitive cells.