Eag K+ Channel Binding to CaMKII: Structural and Biochemical Characterization

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In drosophila, the tetrameric Ether-à-go-go (EAG) voltage-gated K+ channel contains in its C-terminal cytoplasmic region a substrate-like binding domain for Ca2+/calmodulin-dependent protein kinase II (CaMKII-BD). This Ser/Thr kinase forms a dodecamer and each subunit includes an association domain mediating multimerization and a catalytic domain corresponding to the typical protein kinase fold. In between these domains, CaMKII contains a regulatory segment (RS) harboring a CaMKII-BD with homology to that of EAG followed by a Ca2+/CaM-binding region. This RS has a regulatory role, mediating the inhibition and activation of the kinase. CaMKII is activated by Ca2+/CaM-binding and can become Ca2+/CaM-independent upon phosphorylation of its CaMKII-BD. EAG is phosphorylated by CaMKII in vivo, and the two proteins form a stable complex in vitro. CaMKII-bound EAG keeps the kinase active in the absence of Ca2+/CaM and autophosphorylation. The interaction of the constitutively active CaMKII kinase domain with the EAG CaMKII-BD has been characterized. The measurement of KD values showed that the EAG CaMKII-BD is a high affinity CaMKII substrate, lying in the nanomolar range in comparison with the micromolar range of typical protein kinase substrates. Crystal structures were determined for the ternary complexes CaMKII-Mg2+/ATP-EAG and CaMKII-Mg2+/ADP-EAGP corresponding to the initial and final states of the trans-phosphorylation mechanism, respectively. These structures are globally very similar, with relative rearrangements at the catalytic site. An alanine scan of the EAG residues lying at the CaMKII-EAG interface revealed that practically every residue contributes significantly for the binding strength of the complex. The association of the two proteins in the cell might lie beyond the usual transient kinase-substrate association model, likely corresponding to a long-lasting robust complex.