Role of the N- and C-Lobes of Calmodulin in the Activation of Ca²⁺/Calmodulin-Dependent Protein Kinase II

Abstract

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca2+-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater Kact than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca2+-binding to each lobe of CaM during intracellular Ca2+ oscillations.

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

“…The determined binding affinities were in agreement with affinities determined for the full-length enzyme [33]. Ca 2+ /CaM bound to CaMKI and CaMKIV with >20-times higher affinity.…”

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of CaV1.2

“…Fl-A 1588 had a much weaker affinity for each individual EF-hand domain of CaM ( K d of 2.41 ± 0.32 μM for CaM 1–80 and K d of 14.4 ± 6.4 μM for CaM 76–148 ) as compared to full-length CaM 1–148 . Although the N-domain of CaM has been reported to be sufficient to promote initial attachment and activation of CaMKII [45], in most cases where there is a difference between the domains of CaM, it is the interaction of a target with the C-domain of CaM that is more energetically favorable. Thus, the observation that the N-domain of CaM binds to Fl-A 1588 with a 6-fold more favorable equilibrium constant than that of the CaM C-domain is unusual compared to the majority of CaM target interactions.…”

Calcium: Amplitude, Duration, or Location?

“…Simulations showed that the fast binding N lobe of calmodulin is much more sensitive to calcium channel location than the slow binding C lobe, and that saturated calmodulin N lobe itself forms a distinct gradient within the spine. Because calcium binding to N or C lobes of calmodulin differentially activates CaMKII (Forest et al, 2008), this may be a mechanism by which calcium location can affect plasticity.…”