Development and characterization of functional antibodies targeting NMDA receptors

Nami Tajima,Kevin Michalski,Albert Y Lau,Hiro Furukawa,Michael C Regan,Noriko Simorowski,Remy A Yovanno,Ricardo Gómez

doi:10.1038/s41467-022-28559-3

Abstract

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

Highlights

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness
To isolate functional antibodies against the GluN1-GluN2B NMDARs, we immunized mice with purified intact rat GluN1a-GluN2B NMDAR proteins prepared in lauryl maltose neopentyl glycol (LMNG)[40]
We screened for IgGs that showed signal in an enzyme-linked immunosorbent assay (ELISA) using the intact rat GluN1aGluN2B NMDAR proteins in the presence of 0.01% LMNG and no signal in Western blotting executed in a denaturing condition (Fig. 1a)

Summary

Results

Identification and characterization of anti-GluN1-GluN2B NMDAR inhibitory antibodies. To understand whether the inhibitory effect was due to specific binding of the IgG2 or Fab[2] to the GluN2B ATD as observed in the cryo-EM structure or other factors, we conducted site-directed mutagenesis on the interacting residues on the GluN2B subunit and tested the inhibitory effect on the ion channel activity (Fig. 3e, f). The specific residues involved in the interaction between GluN1b-GluN2B NMDAR and Fab[5] were captured by the x-ray crystallographic structure of GluN1b-GluN2B ATDFab[5] at 4.54 Å (Supplementary Fig. 6 and Supplementary Table 1), which shows that the binding involves His[311], Ser[312], Phe[313], Gln[331], Ser[332], Asn[333], and Met[334] of GluN2B in the R1 lobe and residues from CDR1 and CDR3 of the light and the heavy chains of IgG5, respectively.

73 Å M3’M3’

Discussion

Methods