Characterization of CaMKIIα holoenzyme stability

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca2+-dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKII alpha using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T-m = 36 degrees C), which is slightly stabilized by ATP/MgCl2 binding (apparent T-m = 40 degrees C) and significantly stabilized by regulatory segment binding (apparent T-m = 60 degrees C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T-m similar to 90 degrees C), and the holoenzyme structure has multiple unfolding transitions ranging from similar to 60 degrees C to 100 degrees C. Using MP, we compared a CaMKII alpha holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact.