Characterization of Camkiialpha Holoenzyme Stability

Abstract

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation. CaMKII comprises 2% of the total protein in dendritic spines, where it plays important signaling roles allowed for by its unique structure. Each CaMKII subunit is comprised of a kinase domain, regulatory segment, linker region, and hub domain that is responsible for oligomerizing the subunits. Understanding the hub domain and its dynamics (i.e., how it assembles and falls apart) is crucial, and we have pursued experiments to interrogate holoenzyme stability using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability, which is significantly stabilized by regulatory segment binding. The hub domain alone is extremely stable, whereas the holoenzyme structure has multiple unfolding transitions ranging from ∼60°C to 100°C. Using MP, we compared the masses CaMKIIα splice variants with different variable linker regions and determined that the holoenzymes are less stable) compared with the hub domain alone. These results indicate that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. Insight into the dynamics of the holoenzyme gives us a broader understanding of CaMKII activation-triggered subunit exchange. Active CaMKII exchanges subunits with neighboring CaMKIIs and spreads its activation state. This might have a significant role in memory formation where it remains unclear how memories are stored for long periods of time. We interrogated CaMKII subunit exchange using FRET and single-molecule microscopy. Our results indicate that stability correlates with subunit exchange rate. Some instability may be required to initiate this dynamic process. These data provide important insight to holoenzyme biophysics and its role in CaMKII biology.