Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM

CaMKII predominantly assembles into a 12-meric ring assembly, primarily consisting of α and β isoforms in the brain. Previous biochemical studies reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKIIα/β heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employ high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKIIα/β at a 3:1 ratio. Our findings reveal that the α and β subunits are intermixed within the 12-meric ring assembly, with a probability exceeding 83% that β subunits are positioned adjacently. Furthermore, in the activated state, CaMKIIα/β heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKIIβ subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKIIβ subunits within the CaMKIIα/β heterododecamer.