Fluorescence Polarization and Fluctuation Analysis Reveals Covert Changes in CaMKII Holoenzyme Organization Triggered by Calmodulin and Camkiintide

The calcium-calmodulin dependent protein kinase-II (CaMKII) is a multimeric kinase involved in memory and synaptic modulation. In the auto-inhibited state, a CaMKII holoenzyme is composed of catalytic domain pairs that are distributed around a central core of association domains. Upon binding calcium/calmodulin (Ca2+/CaM) the kinase is activated. Ca2+/CaM binding is also thought to trigger a conformational change that exposes the T interaction site on the catalytic domain, The T-site allows the kinase to interact with several synaptic proteins, including NMDA receptors. In the context of the intact holoenzyme, little is known about the conformational changes initiated by Ca2+/CaM binding to the regulatory domain or by subsequent T-site interactions. We have used Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid microscopy technique to assess these changes. FPFA simultaneously measures Forster resonance energy transfer (FRET, a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and diffusion time (an attribute sensitive to the mass and shape of a protein complex). In homogenates, activation with Ca2+/CaM triggered catalytic-domain pair extension (as indicated by diffusion time) without catalytic-domain pair separation (as assayed by homo-FRET). CaMKIINtide inhibitor peptide, a T-site ligand, triggered catalytic-domain pair separation, but only when co-incubated with Ca2+/CaM. Furthermore, a T-site mutant, CaMKII (I205K), which cannot bind to NMDA receptors or translocate to spines, exhibited an increase in FRET (catalytic-domain dimerization) upon activated by Ca2+/CaM, and also prevented CaMKIINtide triggered catalytic-domain pair separation. These observations suggest that catalytic-domains remain paired in the activated holoenzyme by virtue of T-site regulated interactions. Catalytic-domain pairs only separate if exogenous T-site ligands can effectively compete for the sites holding catalytic domains together.