The CaMKII holoenzyme structure in activation-competent conformations

Janette B Myers,K Ulrich Bayer,Steven J Coultrap,Vincent Zaegel,Adam P Miller,Steve L Reichow

doi:10.1038/ncomms15742

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.

Highlights

The Ca2 þ /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
Both forms of molecular memory are enabled by the CaMKII holoenzyme structure, which is assembled by the association of 12 subunits via their C-terminal association domains
In order to enable the intersubunit autophosphorylation at T286, there must be a conformation with a flexible kinase domain positioning that allows for one kinase domain to access the regulatory domain of its neighbour

Summary

Introduction

The Ca2 þ /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. A recent crystallographic model has been described for an artificial CaMKIIa construct in which the variable linker domain was completely deleted (linker-less)[23] After bacterial expression, this linker-less construct formed a compact conformation, in which the kinase/regulatory domains are packed closely against the central hub complex. In order to enable the intersubunit autophosphorylation at T286, there must be a conformation with a flexible kinase domain positioning that allows for one kinase domain to access the regulatory domain of its neighbour Such flexibility could either be induced by Ca2 þ /CaM-stimulation or be an intrinsic property of the activation-competent conformation of a holoenzyme that is present even in its basal state. Any equilibrium must include at least one additional activation-competent conformation in which the Ca2 þ /CaMbinding sites are accessible for holoenzyme activation

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Results

Conclusion