Dual effect of ATP in the activation mechanism of brain Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin

The activation mechanism of Ca2+/calmodulin-dependent protein kinase II (alpha CaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal [Torok, K., and Trentham, D.R. (1994) Biochemistry 33, 12807-12820] is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin [Drum et at. (2000) J. Biol. Chem. 275,363340-36340] (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca2+/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alpha CaMKII as well as with alpha CaMKII-derived calmodulin-binding peptides in the presence of Ca2+. These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alpha CaMKII with estimated interprobe distances of 40 and 22-26 Angstrom. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca2+/TA-cal with alpha CaMKII is estimated as 2 x 10(7) M-1 s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca2+/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M-1 s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.