Fig. 2: GluN1 hinge mutants disrupt the EphB2–GluN1 interaction in HEK293T cells.

a A model of domain organization of the GluN1 and the GluN2B subunits of the NMDA receptor. The red box indicates the N-terminal domain (NTD) of GluN1. b The crystal structure of the NTD of GluN1 (PDB: 4PE5). The red box indicates the hinge region within the NTD with specific amino acids highlighted in red. c The structure of the GluN1 NTD hinge region represented in cartoon form with the location of six amino acids highlighted in red (I272, N273, T335, G336, R337, N350). d The surface representation of the structure of the GluN1 NTD hinge region with the location of the six amino acids in c highlighted in red. e Charge map of the GluN1 NTD hinge region. The charge map was generated using the adaptive Poisson–Boltzmann solver (APBS) plugin in PyMOL. f Charge maps of GluN1 NTD hinge region mutants (WT, Quintuple, Sextuple). Yellow outline indicates the location of the six key hinge region amino acids in WT GluN1. g Representative images of the PLA assay results in HEK293T cells. HEK293T cells were transfected with either WT Myc-GluN1, Quintuple mutant Myc-GluN1 (I272A/N273A/T335A/G336A/R337A), or Sextuple mutant Myc-GluN1 (I272A/N273A/T335A/G336A/R337A/350Q), and GluN2B, FLAG-tagged-EphB2, and EGFP. The upper panels show PLA signal alone. The lower panels are the merged images with EGFP in green and PLA signal in magenta. Scale bar = 10 µm. h Quantification of the effects of GluN1 mutants on PLA puncta number. PLA puncta number are quantified by counting the number of puncta per 100 µm² in EGFP⁺ cells and normalizing to the WT condition (***p < 0.005, ANOVA; green dots represent n = 30 cells for each condition).