CaMKII binds both substrates and activators at the active site

Abstract

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein that is required for successful long-term memory formation. Ca2+/CaM activates CaMKII by binding to its regulatory segment, thereby making the substrate binding pocket available. One exceptional feature of this kinase is that two binding partners have been shown persistently activate CaMKII after the Ca2+ stimulus dissipates. The molecular details of this phenomenon are unclear. Despite having a large variety of interaction partners, the specificity of CaMKII has not been structurally well-characterized. To this end, we solved X-ray crystal structures of the CaMKII kinase domain bound to four different binding partners: a peptide substrate, a substrate/activator, a substrate/binding partner, and an inhibitor (AMPA-type glutamate receptor, NMDA-type glutamate receptor, Tiam1, and Densin-180). We show that all four binding partners use similar interactions to bind across the substrate binding pocket of the CaMKII active site. We generated a sequence alignment based on our structural observations, which revealed conserved interactions across these binding partners. The structures presented here shed much-needed light on the interaction between CaMKII and its binding partners. These observations will be crucial in guiding further biological experiments.

Synaptic memory requires CaMKII

“…The second class of inhibitory peptide is derived from the autoinhibitory domain of CaMKII ( Bayer et al, 2001 ; Leonard et al, 1999 ). Although these inhibitory peptides were thought to bind to separate sites (S and T sites), recent structural studies of the binding of GluN2B and other interacting peptides to CaMKII indicate that these peptides use similar interactions to bind across the substrate binding pocket of the CaMKII active site ( Özden et al, 2020 ). Thus, at present it is not possible to use these two classes of inhibitors to distinguish between the disruption of GluN2B binding and the blockade of kinase activity.…”

Molecular basis of interactions between CaMKII and α-actinin-2 that underlie dendritic spine enlargement

“…A more logical possibility to explain increased actinin-CaMKII association hours after LTP induction is the formation of NMDAR-CaMKII complexes that endure hours following CaMKII activation (Bayer et al, 2001; Yasuda et al, 2022). CaMKII binds tightly to a site centred on S1303 in the GluN2B C-terminal tail (Omkumar et al, 1996; Strack, McNeill, et al, 2000), and recent crystallographic work shows how this sequence wraps around the kinase domain using a binding mode that necessitates full displacement of the regulatory segment from the kinase domain (Ozden et al, 2022). To investigate the possibility that GluN2B supports CaMKIIα binding to α-actinin-2, we compared pull-down of full-length CaMKIIα with magnetic glutathione beads charged with either GST or GST-EF1-4 and determined the effect of including a purified fragment of the GluN2B tail spanning positions 1260-1492 ( Figure 3-figure supplement 1 E) – hereafter referred to as ‘GluN2Bc’.…”

“…The duration of CaMKII activation is prolonged beyond the duration of Ca 2+ elevation by auto-phosphorylation at T286, although pT286 autonomy is reversed by phosphatases including protein phosphatase 1 within ~ 10 s (Yasuda et al, 2022). In complex with NMDARs, the substrate-binding groove of the CaMKII kinase domain is occupied (Ozden et al, 2022), which leaves the regulatory segment accessible to interact with α-actinin-2 ( Figure 6 C). In this way, α-actinin-2 is enriched in dendritic spine heads where it supports spine head enlargement through its ability to crosslink actin filaments via its N-terminal actin-binding domain ( Figure 6 C).…”

Molecular basis of interactions between CaMKII and α-actinin-2 that underlie dendritic spine enlargement

“…Binding of Ca 2+ /CaM to a central regulatory segment releases the segment from the kinase domain enabling access to substrates and interaction partners ( Yasuda et al, 2022 ). The CaMKII kinase domain has many documented substrates and binding partners ( Özden et al, 2020 ). Formation of a highly stable complex between the CaMKII kinase domain and the C-terminal tail of NMDA receptor (NMDAR) GluN2B subunits is thought to be critically important for learning and memory ( Sanhueza et al, 2011 ).…”