Separation of Domain Contacts Is Required for Heterotetrameric Assembly of Functional NMDA Receptors

Abstract

The precise knowledge of the subunit assembly process of NMDA receptors (NMDA-Rs) is essential to understand the receptor architecture and underlying mechanism of channel function. Because NMDA-Rs are obligatory heterotetramers requiring the GluN1 subunit, it is critical to investigate how GluN1 and GluN2 type subunits coassemble into tetramers. By combining approaches in cell biology, biochemistry, single particle electron microscopy, and x-ray crystallography, we report the mechanisms and phenotypes of mutant GluN1 subunits that are defective in receptor maturation. The T110A mutation in the N-terminal domain (NTD) of the GluN1 promotes heterodimerization between the NTDs of GluN1 and GluN2, whereas the Y109C mutation in the adjacent residue stabilizes the homodimer of the NTD of GluN1. The crystal structure of the NTD of GluN1 revealed the mechanism underlying the biochemical properties of these mutants. Effects of these mutations on the maturation of heteromeric NMDA-Rs were investigated using a receptor trafficking assay. Our results suggest that the NTDs of the GluN1 subunit initially form homodimers and the subsequent dimer dissociation is critical for forming heterotetrameric NMDA-Rs containing GluN2 subunits, defining a molecular determinant for receptor assembly. The domain arrangement of the dimeric NTD of GluN1 is unique among the ionotropic glutamate receptors and predicts that the structure and mechanism around the NTDs of NMDA-Rs are different from those of the homologous AMPA and kainate receptors.

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

“…4A). This loop adopts different configurations in various GluN1 NTD crystal structures (11,16) and displays high flexibility in molecular dynamic simulations (25). To explore the possible role of I133 in photo-cross-linking, we generated a series of double mutant receptors, mutating I133 to G, A, V, or L on the GluN1-Y109amb background.…”

“…1A). Besides having essential functions in receptor assembly (10,11), recent studies of the NTDs have also revealed that the individual GluN2 (12)(13)(14) and GluN1 (15) NTDs fine-tune NMDAR gating and pharmacological properties by undergoing large-range conformational changes. The recent X-ray crystal structure of a GluN1/GluN2B NTD complex reveals a unique arrangement of the two NTD protomers with intersubunit interactions distinct from those observed in AMPA and kainate receptors (16,17).…”

The N-terminal Domain Modulates α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Desensitization

“…Many studies have confirmed that NTDdriven dimerization is an important early step in the assembly (Refs. 16,18,59; for reviews, see Refs. [63][64][65], and point mutations in the NTD can lead to impaired maturation and surface expression (18,59).…”

“…16,18,59; for reviews, see Refs. [63][64][65], and point mutations in the NTD can lead to impaired maturation and surface expression (18,59). Nascent receptor dimers held together by N-terminal dimerization and by transmembrane segment packing, but with clearly separate LBDs, have been identified in an electron microscopic analysis of AMPA receptor biosynthesis (16).…”

Mapping the Binding of GluN2B-SelectiveN-Methyl-d-aspartate Receptor Negative Allosteric Modulators

“…These structural data shed further light on differences between the NMDA receptor ATDs and those of kainate and AMPA receptors. Whereas the ATDs of all ionotropic glutamate receptors have a bilobed architecture and consist of two domains (R1 and R2), NMDA receptors are unique compared with kainate and AMPA receptors in that the R1 and R2 lobes are oriented differently with respect to each other (Karakas et al, 2009(Karakas et al, , 2011Farina et al, 2011). This difference in orientation of the R1 and R2 domains results in distinct association between the GluN1/ GluN2B ATDs that differs in molecular detail from non-NMDA receptor ATDs, which can associate as homodimers or heterodimers (Zhao et al, 2012;Kumar et al, 2009Kumar et al, , 2011 and extensive strong interactions between the R1-R1 and R2-R2 domains of the dimer.…”