Structural dynamics of CaMKII activation

Abstract

The ubiquitously expressed calcium/calmodulin dependent protein kinase II (CaMKII) functions as a transducer of calcium (Ca2+) signaling by responding to the amplitude, duration, and frequency of Ca2+ transients. During periods of elevated Ca2+, CaMKII is activated by calcium-calmodulin (Ca2+/CaM) binding. A subsequent autophosphorylation at Thr286 allows for Ca2+-independent activity and endows this enzyme with a conformational memory of prior activation. CaMKII activity is regulated by a myriad of factors including CaM binding, autophosphorylation, and catalytic-regulatory domain interactions referred to as autoinhibition. While these variables have been linked to CaMKII function, the underlying structural and dynamic framework of activation and conformational memory is poorly understood. Here we utilize site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) to explore the conformational changes associated with CaMKII activation and conformational memory. EPR parameters were collected for the regulatory domain where CaM binding and autophosphorylation sites are located. Our results indicate the regulatory domain undergoes significant structural changes between several discrete conformations dependent on autophosphorylation and CaM binding. The CaM binding region is flexible in the apo state but has an induced rigidity in the presence of Ca2+/CaM indicative of a binding event. Investigation of the regulatory domain outside the CaM binding region revealed an increase in protein backbone dynamics with a Thr286Glu autophosphorylation mimic and/or in the presence of Ca2+/CaM. This data provides a structural and dynamic perspective consistent with the current biochemical activation model where CaM binding disrupts autoinhibition by disengaging regulatory and catalytic domains. We predict the enhanced flexibility facilitates Ca2+/CaM binding and may play a role in Ca2+ independent activity. The adjacent regulatory loop showed similar flexibility suggesting this region functions as a hinge between regulatory and catalytic domains allowing for release and reinstatement of autoinhibition.