Journal: Cell Rep / Year: 2018 Title: Structural Basis of the Proton Sensitivity of Human GluN1-GluN2A NMDA Receptors. Authors: Jin-Bao Zhang / Shenghai Chang / Pan Xu / Miao Miao / Hangjun Wu / Youyi Zhang / Tongtong Zhang / Han Wang / Jilin Zhang / Chun Xie / Nan Song / Cheng Luo / Xing Zhang / Shujia Zhu Abstract: N-methyl-D-aspartate (NMDA) receptors are critical for synaptic development and plasticity. While glutamate is the primary agonist, protons can modulate NMDA receptor activity at synapses during ...N-methyl-D-aspartate (NMDA) receptors are critical for synaptic development and plasticity. While glutamate is the primary agonist, protons can modulate NMDA receptor activity at synapses during vesicle exocytosis by mechanisms that are unknown. We used cryo-electron microscopy to solve the structures of the human GluN1-GluN2A NMDA receptor at pH 7.8 and pH 6.3. Our structures demonstrate that the proton sensor predominantly resides in the N-terminal domain (NTD) of the GluN2A subunit and reveal the allosteric coupling mechanism between the proton sensor and the channel gate. Under high-pH conditions, the GluN2A-NTD adopts an "open-and-twisted" conformation. However, upon protonation at the lower pH, the GluN2A-NTD transits from an open- to closed-cleft conformation, causing rearrangements between the tetrameric NTDs and agonist-binding domains. The conformational mobility observed in our structures (presumably from protonation) is supported by molecular dynamics simulation. Our findings reveal the structural mechanisms by which protons allosterically inhibit human GluN1-GluN2A receptor activity.
Mass: 94192.172 Da / Num. of mol.: 2 / Details: 2 mM L-Glutamate / Mutation: E656R, E657R / Source: (gene. exp.) Homo sapiens (human) / Gene: Grin2a / Cell line (production host): HEK293S GnTl- / Production host: Homo sapiens (human) / References: UniProt: Q12879
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Experimental details
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Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction
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Sample preparation
Component
Name: Human GluN1/GluN2A NMDA receptors in the glutamate/glycine bound state at pH 6.3, Class I Type: COMPLEX / Details: with the presence of Glycine,L-glutamate and EDTA / Entity ID: 1, 2 / Source: RECOMBINANT
Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 kelvins Details: blot for 2 seconds before plunging in liquid ethane
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Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Microscope model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELDBright-field microscopy
Image recording
Average exposure time: 12 sec. / Electron dose: 56 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 4
Image scans
Movie frames/image: 40 / Used frames/image: 1-40
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Processing
EM software
ID
Name
Category
1
Gautomatch
particleselection
4
Gctf
CTFcorrection
7
UCSF Chimera
modelfitting
13
PHENIX
modelrefinement
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Number of particles selected: 722287
3D reconstruction
Resolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 193878 / Symmetry type: POINT
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