Structural examination of autoregulation of multifunctional calcium/calmodulin-dependent protein kinase II

Regulation of Ca2+/calmodulin-dependent protein kinase II is likely based o n an auto-inhibitory mechanism in which a segment of the kinase occupies th e catalytic site in the absence of calmodulin, We analyze potential auto-in hibitory associations by employing charge reversal and hydrophobic-to-charg ed residue mutagenesis, We identify interacting amino acid pairs by using d ouble mutants to test which modification in the catalytic domain complement s a given change in the auto-inhibitory domain. Our studies identify the co re pseudosubstrate sequence (residues 297-300) but reveal that distinct seq uences centered about the autophosphorylation site at Thr-286 are involved in the critical auto-inhibitory interactions. Individual changes in any of the residues Arg-274, His-282, Arg-283, Lys-291, Arg-297, Phe-293, and Asn- 294 in the auto-inhibitory domain or their interacting partners in the cata lytic domain produces an enhanced affinity for calmodulin or generates a co nstitutively active enzyme. A structural model of Ca2+/calmodulin-dependent protein kinase II that incorporates these interactions shows that Thr-286 is oriented inwardly into a hydrophobic channel. The model explains why cal modulin must bind to the auto-inhibitory domain in order for Thr-286 in tha t domain to be phosphorylated and why introduction of phospho-Thr-286 produ ces the important Ca2+-independent state of the enzyme.