Calcium/calmodulin-dependent kinase II (CaMKII) is unique among protein kinases because it forms a dodecameric holoenzyme that responds not just to the amplitude but also to the frequency of the activating signal. CaMKII is of central importance in neuronal signaling because it transduces intracellular calcium influx into the phosphorylation of ion channels and changes that alter synaptic strength (Kennedy et al., 1983
; Nairn et al., 1985
; Schulman and Greengard, 1978
). The activity of CaMKII is switched on by calcium spikes, but the enzyme escapes calcium dependence when the calcium spike frequency exceeds a characteristic threshold.
/CaM) activates CaMKII by displacing an inhibitory segment that blocks the active site of the enzyme. This segment is phosphorylated in a frequency dependent manner, and once phosphorylated it no longer blocks the enzyme, even in the absence of Ca2+
/CaM (De Koninck and Schulman, 1998
; Hanson et al., 1989
; Miller and Kennedy, 1986
). In this way the enzyme is capable of storing a molecular “memory” of activating pulse trains, a property responsible for the key role played by CaMKII in the acquisition of long-term potentiation (LTP) (Malenka and Bear, 2004
Each subunit of a CaMKII holoenzyme consists of a Ser/Thr-specific kinase domain followed by a regulatory segment that binds to Ca2+
/CaM (Hudmon and Schulman, 2002
) (). The regulatory segment is followed by a flexible linker of variable length that connects to the hub domain (also referred to as the association domain). The hub domains assemble into two hexameric rings that form the dodecameric holoenzyme (Hoelz et al., 2003
; Rellos et al., 2010
; Shen and Meyer, 1998
CaMKII Subunit Architecture and Activation
Several structures of the isolated kinase domain, determined previously, illustrate how the regulatory segment controls kinase activity (Figure S6
). Three critical phosphorylation sites (Thr 286, Thr 305 and Thr 306) are located within the regulatory segment, which is divided into three regions denoted R1, R2 and R3 (see ). Thr 286 (mouse α isoform numbering) is at the base of an α helix formed by the regulatory segment. This α helix blocks substrate binding in the autoinhibited structure by occupying a hydrophobic groove on the kinase domain. Thr 305 and Thr 306 lie at the heart of the calmodulin binding region of the regulatory segment (the R3 element). Phosphorylation at Thr 286 occurs in trans
, between two kinase subunits of the same holoenzyme (Hanson et al., 1994
), and disrupts the docking of the R1 and R2 elements of the regulatory segment against the kinase domain (). This releases autoinhibition, even in the absence of calcium, thereby conferring calcium-independent activity to the kinase. Phosphorylation of Thr 305 and Thr 306 prevents calmodulin binding (Colbran, 1993
; Hanson and Schulman, 1992
). Studies using transgenic mice show that mutation of Thr 286, a site of phosphorylation in the regulatory segment, results in impaired learning, while mutation of the two other phosphorylation sites (Thr 305 and Thr 306) result in learning that is less adaptable (Elgersma et al., 2002
; Silva et al., 1992
There are four CaMKII genes (α, β, γ, δ) in humans. The enzymes produced by these genes have virtually identical kinase domains (~95% sequence identity) and very similar hub domains (~80% identity). The principal difference between these gene products is in the linker connecting the kinase domain to the hub domain, which is variable in both sequence and length. At least 38 distinct mammalian splice variants are generated by lengthening or shortening this region (Tombes et al., 2003
), and changes in the linker length are correlated with changes in the frequency response of the enzyme (Bayer et al., 2002
). The origin of the coupling between linker length and the frequency response is unclear.
All of the essential features of CaMKII are conserved across metazoans. The regulatory segment and the three phosphorylation sites within it are invariant. The residues that mediate the oligomerization of the hub domain are conserved from representatives of the earliest metazoans, such as hydra and sea urchins, to humans (Figure S1
). This conservation in CaMKII dates to the evolutionary stage when the first synapse was thought to have formed (Ryan and Grant, 2009
). The linker region between the regulatory segment and hub domain is the only major site of variation over the ~1200 million year evolution of this enzyme.
Some information about the quaternary structure of the CaMKII holoenzyme assembly has emerged from electron microscopy (EM) and small angle X-ray scattering (SAXS). In one set of EM reconstructions, individual kinase domains are arranged above and below the midplane of the central hub (Kolodziej et al., 2000
; Woodgett et al., 1984
). In another set of EM reconstructions, the kinase domains occupy radial positions at the midplane of the central hub and are in close proximity to one another (Morris and Torok, 2001
). The radial arrangement of kinase domains in this set of EM reconstructions is similar to that seen in models of CaMKII based on SAXS (Rosenberg et al., 2005
). These disparate views of the holoenzyme structure have not been reconciled.
We now present the crystal structure of the full-length dodecameric human isoform of CaMKII in an autoinhibited state. This structure reveals an unanticipated and very compact arrangement of kinase domains around the central hub. A portion of the regulatory segment, bearing the phosphorylation sites Thr 305 and Thr 306, is incorporated into the tertiary structure of the subunits of the central hub. We present SAXS and biochemical data indicating that the compact conformation of the holoenzyme seen in the crystal is present in solution and that the equilibrium between compact and extended forms is altered by changing the length of the linker. Analysis of a stochastic kinetics computational model for CaMKII shows that alterations in the equilibrium constant between the compact and extended forms of the holoenzyme can alter the frequency response of the enzyme. Based on these observations, we propose that a dynamic equilibrium between compact and extended autoinhibited states modulates CaMKII autoinhibition to set a tunable threshold for the response to calcium spikes.