Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits

Abstract

N-Methyl-D-Aspartate (NMDA) receptors are essential for many brain functions. These receptors are heterotetramers typically comprising two GluN1 subunits and two GluN2 subunits. The latter could alternate among four subtypes (N2A-N2D) and determine the functional diversity of NMDA receptors. For example, receptors containing N2C or N2D exhibit 50-fold lower channel open probability (Po) than those containing N2A. Structures of N2A- and N2B-containing receptors have been extensively characterized, providing molecular basis for understanding NMDA receptor function. Here we report the cryo-EM structures of N1-N2D and N1-N2C di-heterotetramers (di-receptors), and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a resolution up to 3.0 angstroms. Structural analysis showed that the bilobate N-terminal domain (NTD) in N2D adopted an intrinsic closed conformation, leading to a compact NTD tetramer in N1-N2D receptor. Functional studies further demonstrated that, in di-receptors containing N2D but not N2A or N2B, crosslinking NTD at the tetrameric interface had no effect on channel activity, while crosslinking ligand-binding domain (LBD) of two N1 protomers significantly elevated Po. Surprisingly, we found that the N1-N2C di-receptors spontaneously oscillated between symmetric and asymmetric conformation. The later one occupied a predominant population, whereby two N2C protomers exhibited distinct conformation. This asymmetry, which was also found to a lesser extent in N1-N2A di-receptor, was further locked by the binding of an N2C-specific allosteric potentiator PYD-106 to a unique binding pocket between NTD and LBD in only one N2C protomer. Finally, N2A and N2C in the N1-N2A-N2C tri-receptor displayed the conformation close to that found in one protomer of N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse functional properties of major NMDA receptor subtypes.

Bi-directional allosteric pathway in NMDA receptor activation and modulation

“…Under apo conditions, we primarily observe a high FRET state of 0.97 which correlates to a FRET distance of approximately 29 Å. This distance agrees with the published cryo-EM structure of the GluN1/GluN2D receptor bound by glycine and competitive antagonist CPP, where the distance between Phe554 on both GluN1 subunits is approximately 31 Å 29 . This finding is consistent with the GluN1 transmembrane segments in the GluN1/GluN2D receptor being tightly packed and likely representing the closed, inactive channel state of the receptor.…”

“…These differences indicate that the GluN1 and GluN2 subunits occupy distinct conformations. Such differences were previously observed in cryo-EM structures of the GluN1/GluN2C receptor, which display large splaying at the GluN2 subunits and more minor conformational changes at the GluN1 subunits 28, 29 . It is also important to note that the smFRET measurements performed on the GluN1 subunits were acquired at 100 ms bins.…”

“…While recent cryo-EM structures of GluN1/GluN2D show the coupled conformations (compact and super-compact), the decoupled conformations (splayed and super-splayed) have not been observed 28,29 . This is most likely due to the highly dynamic nature of these decoupled states, as observed in our smFRET data, which makes them difficult to classify.…”

“…Amino-terminal domain conformations dictate differences in receptor activation 22, 50 . Decoupling of the amino-terminal domain upon antagonist or inhibitor binding correlates with lower activity of GluN1/GluN2A and GluN1/GluN2B receptors 29, 51 . To test if a similar shift in conformational landscape underlies lower activity of the GluN1/GluN2D receptor, we studied distances across the GluN2 subunit amino-terminal domain in GluN2D- and GluN2A-containing receptors.…”

“…While several cryo-EM structures of the agonist-bound GluN1/GluN2A and GluN1/GluN2D receptors have been solved, the conformational differences underlying their differences in function could not be clearly identified 28, 29, 30, 31, 32 . Additionally, there are currently no apo state structures of the full-length GluN1/GluN2A or GluN1/GluN2D receptors.…”