Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET

Abstract

The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question.

Computationally Discovered Potentiating Role of Glycans on NMDA Receptors

“…Therefore, any processes on the timescale of ~1–100 μs are unlikely to occur in our simulations of GluN1 and GluN2B LBDs. This work does not attempt to address dynamics on the millisecond and longer timescales414243. Finally, the degree of uncertainty introduced by the use of specific models can be estimated from Table 1, which shows that the timescales of opening/closing in GluN1 and GluN2B LBDs predicted by several models are qualitatively the same.…”

NMDA receptors: linking physiological output to biophysical operation

“…The resulting model, predicts correctly that in subsaturating glycine concentrations synaptic-like currents decay faster, and display deeper desensitization during high frequency stimulation 88 . The glycine binding site is located on the LBD of GluN1 subunits 117,121 , where it may serve to stabilize cleft-closed conformation, as illustrated by functional measurements and atomic simulations with isolated GluN1 LBDs 122–124 . However, similar investigations on tetrameric receptors will be necessary to elucidate whether glycine occupancy affects receptor activity by changing the glutamate-binding site or by influencing the gate independently.…”

Single Molecule Data Analysis: An Introduction

“…There is a broad literature, including reviews [207][208][209], on change-point algorithms relying on the AIC [210][211][212][213], the BIC [178,211,212,[214][215][216][217], generalizations of the AIC [201,204] and BIC [37,218], wavelet transforms [219][220][221] and related techniques [222][223][224][225][226].…”

“…The idea here is not only to penalize both the number of change-points detected but also to discretize the number of intensity levels (states) sampled in an smFRET trajectory. STaSI has subsequently been used to explore equilibrium transitions among N-methyl-D-aspartate receptor conformations by monitoring the distance across the glycine bound ligand binding domain cleft [218].…”