The CaMKII holoenzyme structure in activation-competent conformations

Abstract

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.

Variation in assembly stoichiometry in non‐metazoan homologs of the hub domain of Ca2+/calmodulin‐dependent protein kinase II

“…(a)]. Hub domains oligomerize into donut‐shaped assemblies of 12 or 14 subunits, leading to holoenzymes in which the kinase domains are arranged around the assembled hub [Fig. (b)].…”

“…N‐terminal kinase domains (shown in blue) extend outward from the core, tethered to the hub by a flexible linker. The holoenzyme depicted here is a dodecamer, but tetradecameric holoenzymes can be formed as well . In this diagram, which represents the activated state of CamKII, the autoinhibitory regulatory segments (yellow) are shown displaced from the kinase domains.…”

“…Crystal structures of mammalian CaMKII hubs have revealed both tetradecameric forms (composed of seven vertical dimers, PDB codes 1HKX, and 2W2C 12 ) and dodecameric forms (six vertical dimers, PDB codes 5IG3, 3SOA, and 2UX0). Electron microscopic images of negatively stained samples of intact, full‐length human CaMKII‐α show both dodecameric and tetradecameric forms . The oligomerization state of the mouse CaMKII‐α hub has been observed to change from dodecameric to tetradecameric forms in response to the removal of the kinase domains, either through gene truncation or proteolytic cleavage …”

CaMKII regulates the depalmitoylation and synaptic removal of the scaffold protein AKAP79/150 to mediate structural long-term depression

“…CaMKII autonomy was generated by pre-phosphorylating the kinase at Thr-286 by incubating 200 nM CaMKII␣ subunits in 50 mM PIPES, pH 7.0 -7.2, 1 nM BSA, 10 mM MgCl 2 , 100 M ATP, 1 mM CaCl 2 , and 2 M CaM and incubating on ice for 10 min (7,21,75,76). The reaction was stopped by the addition of PIPES dilution buffer containing 5 mM EGTA.…”

Calcium/calmodulin‐dependent kinase II and memory destabilization: a new role in memory maintenance

“…; Hell ; Myers et al . ). During resting state, this enzyme is inactive because of blocking of the substrate binding site (S‐site) and the catalytic domain, both found in the kinase domain, by the pseudosubstrate segment in the regulatory domain (Braun and Schulman ; Hell ).…”