Functional determinants in the autoinhibitory domain of calcium/ calmodulin-dependent protein kinase II: Role of His²⁸² and multiple basic residues

Important determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II (CaMK-II), corresponding to residues 281-302 of the kinase α-subunit sequence, were identified. Replacement of Thr²⁸⁶ with Ala (CaMK-(281-302 Ala²⁸⁶)) had no effect on either the potency (IC₅₀ = 2 μM) or inhibitory mechanism (competitive with ATP) using the catalytic fragment of CaMK-II. Single replacement of charged residues in CaMK-(281-302, Ala²⁸⁶) identified His²⁸², Arg²⁸³, Lys²⁹¹, Arg²⁹⁷, and Lys²⁹⁸ as important determinants (> 10-fold increase in IC₅₀) for potent inhibition of CaMK-II. Glu²⁸⁵, Asp²⁸⁸, Lys²⁹¹, Arg²⁹⁶, and Lys³⁰⁰ were not as essential (<4-fold change in IC⁵⁰) for potent CaMK-II inhibition. Replacement of either Arg²⁸³, Lys²⁹¹, or Arg²⁹⁷, and Lys²⁹⁸ with Ala did not alter the ATP-competitive mechanism of inhibition although the K_i values increased 16-530-fold. However, replacement of His²⁸² with Ala decreased the IC₅₀ by 20-fold and altered the mechanism of inhibition to noncompetitive with respect to ATP. The non-protonated form of His²⁸² was functionally active since decreasing the pH from 7.5 to 5.5 increased the IC₅₀ of CaMK-(281-302, Ala²⁸⁶) almost 20-fold. Histidine protonation also appeared to disrupt the autoinhibitory domain of intact forms of CaMK-II since preincubation of non-proteolyzed rat brain CaMK-II with calcium/calmodulin (in the absence of ATP) at pH 5.5 generated up to 16% calcium-independent activity when assayed at pH 5.5. Similarly, the level of calcium-independent activity of a baculovirus-expressed Asp²⁸⁶ mutant CaMK-II ((D²⁸⁶)mCaMKα) increased to almost 80% calcium independence when assayed at pH 5.5 compared to only 20% when assayed at pH 7.5. The levels of calcium-independent activity of both the (D²⁸⁶)mCaMKα (at pH 5.5 and 7.5) and the rat brain CaMK-II (at pH 5.5) were sensitive to the concentrations of both ATP and peptide substrate (syntide-2) in the assays. These data suggest that the basic residues Arg²⁸³, Lys²⁹¹, Arg²⁹⁷, and Lys²⁹⁸ are important for potent inhibition of CaMK-II and that the non-protonated form of His²⁸² may play a unique role in the ATP-directed mechanism of inhibition by the CaMK-II autoinhibitory domain.