C&SP21: Functional significance of the dynamics of AMPAR extracellular region

A. Collaborating Investigators: Ingo H. Greger,1 Ivet Bahar,2 Tom M. Bartol,3 Terry J.Sejnowski2

B. Institutions: 1MRC Lab of Molecular Biology, Cambridge, UK; 2Pitt, 3Salk

C. Funding Status of Project: MRC Research Institute Cambridge Core Funds (2003 - ) (Greger), BASAL BODYSRC Synaptic Role of the AMPA receptor N-terminal domain (Greger) 4/2016-4/2019



Fig VIII.4 Comparison of AMPAR and NMDAR tetrameric structures. Both structures are composed of three layers: NTD, LBD and TMD/ The dashed cyan line indicates the membrane interface. The extracellular region (NTD + LBD) of NMDAR is more tightly packed than that of AMPAR (from Dutta et al., 2015)

D. Biomedical Research Problem

Ionotropic glutamate receptor (iGluRs) are ligand-gated ion channels that allow for the flow of cations into the postsynaptic cell in response to glutamate binding, thus regulating neurotransmission upon depolarization of the cell membrane. Among iGluR subfamilies, AMPAR and NMDAR play a key role in learning and memory, and in particular the AMPAR is essential to rapid neurotransmission and synaptic plasticity.61 It preferentially functions as a heterotetramer, composed of subunits GluA1 to GluA4, with GluA2 playing a dominant role in neurosignaling. The last two years have seen an explosion in the number of intact (tetrameric) structures resolved for AMPAR and NMDAR; yet the intact structure of heterotetrameric GluA2-containing AMPAR has been elusive. A major breakthrough in the field has been the resolution of such a cryo-EM structure, that of intact GluA2/3 heteromer, by the Greger lab.62 Strikingly, the N-terminal domain (NTD) of the two Glu2/3 dimers in this heterotetramer adopts a compact 'O' shape when viewed from the top (from the ECR) (as opposed to the common 'N' shape of GluA2 homo-tetramer63), and the NTD layer is vertically compressed to make closer contacts with the ligand-binding domain (LBD), reminiscent of subunit packing64,65 in heterotetrameric NMDAR (Fig VIII.4). The NTD and LBD form the ECR of the AMPAR. Their close association suggests that the NTD may play a key role in communicating signals to the transmembrane domain (TMD) and allosterically modulate the TMD gating. Furthermore, ECR flexibility may facilitate the interactions with auxiliary subunits essential for synaptic plasticity (e.g. Fig VIII.5). This C&SP aims at exploring these hypotheses.



Fig VIII.5. ANM of the whole GluA2 AMPAR shows global bending motions that could bring the NTD into proximity with auxiliary subunit TARPs (from Krieger et al., 2015)

E. Methods and Procedures. The Greger and Bahar labs have been productively collaborating in recent years on AMPAR dynamics, first using the NTD dimer structures1,66 and more recently the intact tetrameric structures.2,3 These studies demonstrated that the NTD domains exhibit structural flexibilities comparable to those of AMPAR NTDs. Furthermore, the global modes of motions predicted by ANM67,68 (or ProDy54) revealed the propensity of homotetrameric AMPAR to assume more compact forms similar to NMDARs. The validity of these modes of motions were confirmed by cross-linking experiments between NTD sites predicted by ANM to come into close proximity.2 In the new term, we will first adopt ANM-based analysis to characterize the mode spectrum of the heterotetrameric AMPAR. ProDy analysis already revealed that the O ↔ N transition is enabled by a global ANM mode.62 We will characterize thoroughly the whole spectrum of motions and generate the energy landscape of Glu2/3 heterotetramer, using the recently introduced extension of coMD.69,70 Then we will focus on the ECR motions that induce a pore opening (or cooperative twisting) at the TMD and analyze the conformational events that enable the allosteric coupling between the ECR and the TMD with the help of accelerated MD simulations. 71 In the next phase, we plan to examine the significance of GluA2/3 ECR flexibility in adapting to its interactions with auxiliary proteins such as cornichon homologs,72 TARPs73 or in forming clusters, which will be further tested/validated with structural and single-particle tracking methods in the Greger lab.


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