| Calcium/calmodulin-dependent protein kinase-II (CaMKII) is unique amongst protein kinases for its dodecameric assembly and its complex response to calcium (Ca2+). In this thesis I first explore the history and biological context of this important enzyme. In the first section, I discuss my work towards a better understanding of the oligomeric state of the protein. Single particle analyses of electron micrographs have suggested previously that the holoenzyme forms a dodecamer that contains two stacked 6-fold symmetric rings. In contrast, a recent crystal structure of the isolated association domain of mouse CaMKIIα has revealed a tetradecameric assembly with two stacked 7-fold symmetric rings. I have determined the crystal structure of the C. elegans CaMKII association domain and it too forms a tetradecamer. I also show by electron microscopy, that in its fully assembled form the CaMKII holoenzyme is a dodecamer, but without the kinase domains the association domains form a tetradecamer. I speculate that the holoenzyme is held in its 6-fold symmetric state by the interactions of the N-terminal ∼1-335 residues and that the removal of this region allows the association domain to convert into a more stable 7-fold symmetric form. In the next section, I discuss the crystal structure of the auto-inhibited kinase domain of CaMKII, determined at 1.8 Å resolution. It reveals an unexpected dimeric organization in which the calmodulin responsive regulatory segments form a coiled-coil strut that blocks peptide and ATP binding to the otherwise intrinsically active kinase domains. A threonine residue in the regulatory segment, which when phosphorylated renders CaMKII calmodulin-independent, is held apart from the catalytic sites by the organization of the dimer. This ensures a strict Ca 2+ dependence for initial activation. The structure of the kinase dimer, when combined with small angle X-ray scattering data for the holoenzyme, suggests that inactive CaMKII forms tightly packed auto-inhibited assemblies that convert upon activation into clusters of loosely tethered and independent kinase domains. Finally, I conclude with a discussion of the biological and medical significance of my work and a view to future experiments inspired by this study. |
