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 CaMKIIα and CaMKIIβ variants in the brain. Previous biochemical studies have 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 employed high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKIIα/β at a 3:1 ratio. Our findings revealed that the CaMKIIα and CaMKIIβ subunits are intermixed within the 12-meric ring assembly, with more than 83% probability that CaMKIIβ subunits adjacent to one another. 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.