Real-time PCR was used to amplify the mRNA levels

ational changes of helix aD and the aD-aE loop and hence partial activation of the kinase. It was also reported that CaMKI297 is constitutively active albeit with a relatively low activity. CaMKI297 contains all the residues that form helix aR1 in the apo CaMKI320 and the CaMKI320-ATP and CaMKI315-ATP complexes but its activity is not completely inhibited, suggesting that the CaM-binding segment might play some role in facilitating the autoinhibitory segment in the inhibition of the activity. In the rat CaMKI320, the CaM-binding segment forms a long loop that curves into the entry of the ATP-binding site followed by a short aR2 helix that interacts with the N lobe of the kinase . Particularly, Lys300 of the aR1-aR2 loop forms a salt bridge with the strictly conserved Glu102 of the hinge region, which might prohibit Glu102 from binding ATP or the substrate. Intriguingly, in the CaMKI320-ATP complex, the CaM-binding segment mainly forms a long aR2 helix which protrudes away from the catalytic core. A detailed analysis indicates that helix aR2 of this conformation plays an important role in the maintenance of an inactive state of the enzyme through interaction with Glu102 and stabilization of the inactive conformations of helices aR1 and aD. Specifically, Lys300 on helix aR2 also forms a salt bridge with Glu102; this interaction does not abrogate the ability of Glu102 to bind ATP as Glu102 still makes hydrogenbinding interactions with the 29- and 39-hydroxyls of the ribose moiety of ATP, however, it could have an impact on its ability to bind the substrate as Glu102 is also suggested to play a role in the recognition and binding of Arg at P of the substrate . In addition, the N-terminal part 8 Structures of Human CaMKIa of helix aR2 would have steric conflicts with the C-terminal part of helix aD in the CaMKI293-ATP complex, preventing helix aD from adopting an active conformation. Furthermore, the side chain of Gln305 of aR2 forms two hydrogen-bonding interactions with the side chains of Ser291 and Lys295, and thus helix aR2 also contributes to stabilization of helix aR1 in the inactive conformation. To better understand how CaM binds to and activated CaMKI, we superposed the available structures of kinases with the CaMbinding and/or autoinhibitory segments including other CaMK members and the death-associated protein kinase. In the crystal structure of CaM in complex with a peptide corresponding to the CaM-binding segment of CaMKI, the peptide forms a long a-helix. The NMR spectra of CaM bound to either CaMKI320 or a similar peptide were virtually identical, indicating that the binding mode observed in the CaM-CaMKI peptide complex might be retained in the binding of CaM with CaMKI. Superposition of the CaMKI320-ATP structure with the CaMCaMKI peptide structure and the recently reported CaMKIId-CaM structure based on the CaM-binding segment demonstrates that CaM binds to CaMKI and CaMKII in a similar mode, and helix aR2 in CaMKI320-ATP encompasses almost all the residues required for direct interaction with CaM. Therefore, the position and conformation of the CaM-binding segment in CaMKI320-ATP correspond to a biologically relevant state of CaMKI ready for CaM binding. On the other hand, a short AZ-6102 region at the N-terminus of CaM appears to have steric conflicts with helix aD in the CaMKI320-ATP complex, indicating that proper conformational change or dissociation of the N-terminal part of helix aR2 and the autoinhibitory segment is require