Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly.

Olivia R Buonarati,Steve L Reichow,K Ulrich Bayer,Adam P Miller,Steven J Coultrap

doi:10.1016/j.celrep.2021.110168

Olivia R Buonarati, Steve L Reichow + Show 3 more

Open Access

https://doi.org/10.1016/j.celrep.2021.110168

Copy DOI

Abstract

SUMMARYNeuronal CaMKII holoenzymes (α and β isoforms) enable molecular signal computation underlying learning and memory but also mediate excitotoxic neuronal death. Here, we provide a comparative analysis of these signaling devices, using single-particle electron microscopy (EM) in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assemble mainly as 12-mers (but also 14-mers and even 16-mers for the β isoform). CaMKIIα and β isoforms adopt an ensemble of extended activatable states (with average radius of 12.6 versus 16.8 nm, respectively), characterized by multiple transient intra- and inter-holoenzyme interactions associated with distinct functional properties. The extended state of CaMKIIβ allows direct resolution of intra-holoenzyme kinase domain dimers. These dimers could enable cooperative activation by calmodulin, which is observed for both isoforms. High-order CaMKII clustering mediated by inter-holoenzyme kinase domain dimerization is reduced for the β isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.

Highlights

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of long-term plasticity at excitatory glutamatergic synapses in the hippocampus that is required for learning and memory (Bayer and Schulman, 2019; Hell, 2014; Lisman et al, 2012)
CaMKIIb holoenzymes were expressed in eukaryotic cells (Sf9), purified by chromatographic methods and prepared for negative stain electron microscopy (NSEM) using the same protocols previously described for CaMKIIa
Isolated holoenzymes show no signs of proteolytic degradation by SDS-PAGE (Figure 1B), and CaMKIIb specimens produced well-resolved assemblies resembling the same ‘‘flower-like’’ appearance of CaMKIIa holoenzyme structures observed by NSEM (Figures 1C and 1D)

Summary

Introduction

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of long-term plasticity at excitatory glutamatergic synapses in the hippocampus that is required for learning and memory (Bayer and Schulman, 2019; Hell, 2014; Lisman et al, 2012). Both synaptic plasticity and excitotoxic cell death require the 12-meric CaMKII holoenzyme structure for at least two key regulatory functions: (1) autophosphorylation at T286 (pT286) (Cook et al, 2021; Coultrap et al, 2014; Deng et al, 2017; Giese et al, 1998), which occurs between subunits within a holoenzyme (Hanson et al, 1994) and enables detection of stimulation frequency (De Koninck and Schulman, 1998), and (2) binding to the NMDA-type glutamate receptor subunit GluN2B (Barria and Malinow, 2005; Buonarati et al, 2020; Halt et al, 2012), which requires the holoenzyme structure (Bayer et al, 2006; Strack et al, 2000) and mediates CaMKII accumulation at synapses during long-term potentiation (LTP) and excitotoxic insults (Bayer et al, 2001; Buonarati et al, 2020; Halt et al, 2012) Both pT286 and GluN2B binding require an initial stimulus by Ca2+/CaM but maintain partial ‘‘autonomous’’ kinase activity even after Ca2+/CaM has dissociated (Bayer et al, 2001; Bayer and Schulman, 2019; Miller and Kennedy, 1986). This aggregation requires ischemia-related conditions (such as low pH and higher ADP than ATP concentration) and mediates the extra-synaptic clustering in response to excitotoxic stimuli (Dosemeci et al, 2000; Hudmon et al, 1996, 2001; Vest et al, 2009) but may contribute to the synaptic accumulation in response to LTP stimuli (Hudmon et al, 2005)

Results

Discussion

Conclusion