Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-d-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein

Abstract

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.

Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor

“…Tyrosine phosphorylation of CTD2 by Src kinase is known to alleviate Zn 2ϩ inhibition, but nothing is known about how the CTD modulates gating. We showed that Src phosphorylation increased R H , indicating polypeptide expansion (19). Thus, we reasoned that alterations in the underlying dynamics of this disordered region were involved in allosteric regulation of GluN2B-containing receptors.…”

“…6D). Unlike wild type CTD2 (19), the higher FRET state was still predominant even after phosphorylation. Proline depletion does not prevent the polypeptide expansion induced by Src phosphorylation.…”

“…Src phosphorylation of CTD2 led to a general expansion of the polypeptide, which can be observed as a shift to low FRET efficiency (19). The proline-depleted construct 101 also showed an increased sampling of lower FRET states (Fig.…”

“…The His 6 fusion was purified under reducing and denaturing conditions as described (18,19). The protein was exchanged to nondenaturing conditions by extensive washing while bound to nickelnitrilotriacetic acid-agarose.…”

“…To avoid complications with aggregation, all (6). Previously, we showed that Src phosphorylation causes a uniform expansion of CTD2 (19). Thus, the conformational dynamics of CTD2 may be important in allosteric regulation of GluN2B-containing NMDA receptors.…”