BPS2026 - Adjacent β subunits in CaMKIIα/β hetero-oligomers enable sustained activation and F-actin binding revealed by HS-AFM and mathematical modeling

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) plays a key role in learning and memory and forms a dodecameric holoenzyme. In neurons of the brain, the α and β isoforms are highly expressed. Previous biochemical studies reported that the ratio of α and β isoforms differs among brain regions, suggesting that the stoichiometry of isoforms contributes to the regulation of brain function. However, it has remained unclear whether the holoenzyme is formed as a homo-oligomer or as a hetero-oligomer at the single-molecule level, and how the hetero-oligomer functions. In this study, we co-expressed α and β in mammalian cells, purified the holoenzymes, and directly visualized their structural dynamics at the single-molecule level using high-speed atomic force microscopy (HS-AFM) under a 3:1 α/β ratio that mimics the forebrain composition. Our results showed that α and β subunits are intermixed within the 12-mer. Moreover, β subunits were positioned adjacent to one another with more than 83% probability. To examine the functional role of this arrangement, we constructed a mathematical model to evaluate its binding ability to F-actin. In this model, the adjacent arrangement showed 22 times higher binding affinity than that in a dispersed β-subunit arrangement. Thus, this analysis reveals that their adjacency plays a more important role in binding affinity than the number of β-subunits. Furthermore, HS-AFM observations using mutants suggested that this adjacent arrangement of β subunits extends the duration of CaMKII activation. These findings provide direct evidence for the formation of CaMKIIα/β hetero-oligomers and suggest a structural mechanism linking subunit organization to synaptic plasticity.