CaMKII Binding to GluN2B Is Differentially Affected by Macromolecular Crowding Reagents

Abstract

Binding of the Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B controls long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. Regulation of this interaction is well-studied biochemically, but not under conditions that mimic the macromolecular crowding found within cells. Notably, previous molecular crowding experiments with lysozyme indicated an effect on the CaMKII holoenzyme conformation. Here, we found that the effect of molecular crowding on Ca2+/CaM-induced CaMKII binding to immobilized GluN2B in vitro depended on the specific crowding reagent. While binding was reduced by lysozyme, it was enhanced by BSA. The ATP content in the BSA preparation caused CaMKII autophosphorylation at T286 during the binding reaction; however, enhanced binding was also observed when autophosphorylation was blocked. Importantly, the positive regulation by nucleotide and BSA (as well as other macromolecular crowding reagents) did not alleviate the requirement for CaMKII stimulation to induce GluN2B binding. The differential effect of lysozyme (14 kDa) and BSA (66 kDa) was not due to size difference, as both dextran-10 and dextran-70 enhanced binding. By contrast, crowding with immunoglobulin G (IgG) reduced binding. Notably, lysozyme and IgG but not BSA directly bound to Ca2+/CaM in an overlay assay, suggesting a competition of lysozyme and IgG with the Ca2+/CaM-stimulus that induces CaMKII/GluN2B binding. However, lysozyme negatively regulated binding even when it was instead induced by CaMKII T286 phosphorylation. Alternative modes of competition would be with CaMKII or GluN2B, and the negative effects of lysozyme and IgG indeed also correlated with specific or non-specific binding to the immobilized GluN2B. Thus, the effect of any specific crowding reagent can differ, depending on its additional direct effects on CaMKII/GluN2B binding. However, the results of this study also indicate that, in principle, macromolecular crowding enhances CaMKII binding to GluN2B.

The CaMKII holoenzyme structure in activation-competent conformations

“…A caveat to this previous study, however, is that lysozyme has been shown to bind directly to Ca 2+ /CaM30, and, therefore, potentially complicates the interpretation of these results, especially regarding effects on the apparent EC 50 . Thus, we first compared the effects of molecular crowding by 150 mg ml −1 lysozyme and the putatively more inert molecule bovine serum albumin (BSA) (Fig.…”

Live imaging of endogenous Ca2+/calmodulin‐dependent protein kinase II in neurons reveals that ischemia‐related aggregation does not require kinase activity

“…; Goodell et al . ). The quantified amount of CaMKII in the pellet was normalized, with the aggregation in presence of ADP (≥ 100 μM) without inhibitors in the same experiment set to 100.…”

DAPK1 Mediates LTD by Making CaMKII/GluN2B Binding LTP Specific

“…Cell culture, transfections, protein preparations, and Western analysis were done as previously described (Barcomb et al, 2013; 2014; 2015; Bayer et al, 2001; 2006; Coultrap and Bayer, 2012b; 2014; Goodell et al, 2016; 2014; O'Leary et al, 2011; Singla et al, 2001; Vest et al, 2007) and are detailed in the Supplemental Experimental Procedures. Antibodies and dilutions are shown in Supplemental Table 1.…”

“…In vitro protein binding experiments were done as previously described (Goodell et al, 2014) using immobilized GST-GluN2Bc on sepharose or magnetic beads. DAPK1 constructs (varying concentrations or 1 µM for comparison between constructs) were bound in either EGTA or varying Ca 2+ /CaM as indicated.…”