Fig. 1: Glutamate alone triggers two sequential steps of multidomain rearrangement.
a Representation of SNAP_GluN1-1a/GluN2B fusion protein construct (C-termini are present but not shown; Protein Data Bank accession 3KZZ [https://doi.org/10.2210/pdb3KZZ/pdb] and 5IOU [https://doi.org/10.2210/pdb5IOU/pdb]). SNAP tags are randomly labeled by a mixture of donor (green) and acceptor (red) synthetic dyes. smFRET conformational readout of NMDAR ATD interdimer distance is obtained from receptors with one donor and one acceptor dye. b CGP is a neutral GluN1 ligand and mimics the Apo state of the receptor. GluN1-1a(F484A) and GluN2B(H486A) mutations decrease glycine and glutamate affinity by ~6000× and ~300×, respectively56,71. Each histogram n = 5 movies, SEM error bars. c Glutamate binding leads to increased ATD interdimer distance in two discrete steps. Representative donor (green) and acceptor (red) intensity traces and smFRET traces (blue) in the absence (top), intermediate 100 nM (middle), and saturating 1 mM (bottom) glutamate concentration. In all, 3 µM CGP prevents binding by trace glycine. d smFRET histograms in the presence of various glutamate concentrations. Blue, red, and black lines show global 3-component Gaussian fit of low, intermediate, and high FRET states, respectively. Green line shows the sum of all three components. Each concentration n = 5 movies, SEM error bars. e Glutamate dose–response curve in 3 µM CGP (inset cartoon depicts CGP holding GluN1 LBD open and glutamate occupancy of GluN2B LBD depending on concentration; n = 5 movies, SEM error bars). Low FRET Gaussian fit peak taken as a measure of the activated state. h Hill coefficient.
