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

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 packing^64,65 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.

E. Methods and Procedures. The Greger and Bahar labs have been productively collaborating in recent years on AMPAR dynamics, first using the NTD dimer structures^1,66 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 ANM^67,68 (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.

C&SP20: Structural plasticity of chaperonins determined by cryo-electron microscopy: Modeling the machinery of GroEL and TRiC

A. Collaborating Investigators: Wah Chiu,¹ and Ivet Bahar

B. Institutions: ¹Baylor College of Medicine, ²University of Pittsburgh

C. Funding Status of Project: P01NS092525 (Chiu, Co-PI) 4/1/2016-3/31/2021; 5P41-GM103832-31 (Chiu) 12/1/96-12/31/19

D. Biomedical Research Problem

Chaperonins are essential mediators of many functions in the cell, including protein folding. These are oligomeric machines, of ~ 1 megadalton, with two back-to-back rings enclosing a central cavity that accommodates polypeptide substrates. The Chiu lab has solved near-atomic resolution cryoEM structures of GroEL from bacteria,³⁷ Mm-Cpn from archaea,³⁸ and TRiC from eukaryotes.³⁹ In addition, they have studied them in different nucleotide-binding states and with a variety of substrates.^40-44 These studies show that chaperonins assume various conformations under different conditions, i.e. structural plasticity is an inherent property of chaperonins.

Recent advances in direct electron detectors used in cryoEM^45,46 have made cryoEM a feasible imaging modality to record images of molecular machines with high quantum detection efficiency and high information content. Fig VIII.3 panel a is a 3.7 Å cryoEM map of GroEL using 40,000 particle images showing unambiguous resolution of backbone and side-chain densities (panel b). The time from data collection to a complete model was less than 3 weeks (Roh and Chiu, unpublished). Using a more advanced image processing method known as focused 3D classification,⁴⁷ we re-analyzed each of the 14 protein subunits in each of the GroEL particle images. We were able to retrieve three types of conformations (Figure VIII.3 c), and determined that they occur randomly in each GroEL machine in variable proportions. Each of them has a characteristic apical domain structure, which coincide with different subunit structures in the PDB (chains B, J and I of PDB ID 1XCK). These findings demonstrate the power of cryoEM to reveal variable conformations of protein components in one single biological machine at a time at near-atomic resolution. A structure-based computational analysis of the dynamics of GroEL as well as TRiC can help further our understanding of the structural mechanisms underlying their function and provide insights into the role of structural plasticity in enabling their allosteric machinery.

E. Methods and Procedures. Our ongoing activity is to determine GroEL cryoEM structure with GroES and carboxylase substrate. In our newly funded program project, we will also pursue the cryoEM characterization of TRiC complexed with mutant huntingtin exon extracted from Huntington's disease (HD) cell model and mouse neuron model. These experiments will provide a mechanistic understanding of how TRiC prevents the progression of aggregation in polyQ,⁴⁴ and mutant huntingtin exon⁴⁸ as part of our effort to develop a TRiC-like reagent for HD therapeutics. The Bahar lab has experience on the machinery and conformational variability of GroEL-GroES,^49-53 which will be further analyzed in the light of the new data generated by the Chiu lab, and with the help of novel extensions of elastic network models (ENMs) developed in TR&D1 subaim 1.1. The computationally predicted dynamics will be compared to the structural variability observed in cryoEM, and the interactions that modulate GroEL allosteric machinery will be identified. Similar methods will be used for TRiC, toward elucidating its spectrum of motions and how its dynamics relates to its chaperoning activities. Target sites whose perturbation may alter/control TRiC machinery will be identified using ProDy tools,⁵⁴ toward assisting in the design of new therapeutic strategies against HD. We anticipate a productive interaction between this project and C&SP16 on the identification of neuroprotectives for HD with Dr. Friedlander (UPMC), an expert in the etiology of HD as well as discovery of neuroprotectives against HD.^55-60

C&SP19: Mapping the Supramolecular Organization of Dendritic Spines to Model the Regulation of Synaptic Signal Transfer

A. Collaborating Investigators: Mark H. Ellisman,¹ Andreas Herz,²Terry Sejnowski,³ Tom Bartol³

B. Institutions: ¹University of California, San Diego, ²Ludwig-Maximilians U, Munich, and ³Salk

C. Funding Status of Project: NIDA R01-DA038896-02 ‘Deciphering the dynamical multi-scale structure-function of dendritic spines (Ellisman) 7/1/14 – 6/30/19

D. Biomedical Research Problem

Synapses are the sites of communication between neurons and most excitatory synapses occur onto specialized structures called dendritic spines.³⁴ Generally, spines consist of a spine head and a thin spine neck. The role of spines in influencing electrical and biochemical flow at the synapse is still debated; however, spines can assume a variety of shapes and sizes, and it has recently been shown that spine size is highly correlated with synaptic activation history and network function.¹⁰ Spines are filled with a high concentration of filamentous actin, but the density of actin can vary within and across brain regions.³⁵ The role of the actin network in spine function has not been well studied. The highly branched filamentous actin carries electrostatic charges, which causes ions to condense in its vicinity. Within the condensation profile, known as the electrical double-layer, cations, the main charge carriers for depolarizing phenomena, should propagate with great ease due to the high local concentration. Thus, a significant fraction of the current within the volume of the spines is constrained to flow in nano-slits along charged structures. The multiscale modeling of this process is to be explored in this collaboration.

The Ellisman lab is generating high-resolution (~5 nm) serial EM tomographic volumes of entire spine heads from cerebellum, hippocampus, and striatum. The tissue is prepared using high pressure freezing / freeze substitution to optimally preserve actin network structure and membrane profiles.³⁶ Multi-tilt acquisition and direct electron detection further improve image quality for subsequent feature extraction. Using these datasets, the Herz lab is building spatially accurate 3D models of ionic current propagation in topologically consistent segmentations of the filamentous actin network (Fig VIII.2). These models will be used to perform simulations of reaction-diffusion dynamics using MCell, and multiscale simulations of electro-diffusion dynamics, while enforcing consistent biophysical boundary conditions and accurate ion-channel kinetics.

E. Methods and Procedures

Aim 1: Create a multiscale model. Models will be generated using CellBlender that will include all relevant actors (spine apparatus, postsynaptic density) necessary to computationally reconstitute synapses and explore dynamics of Ca²⁺ and other molecules within spines and spine necks with MCell.

Aim 2: Determine role of actin network in spines of various sizes/strengths. Since spine size is strongly correlated with synaptic strength, we will study differences in actin morphology and its influence on diffusion in spines of various sizes. We will choose spines in the CA1 stratum radiatum that are representative of large (>0.1um3), average (0.02-0.04 um3), and small (<0.01um3) spines. MCell will be used to model diffusion in the CellBlender models created in Aim 1.

Aim 3: Compare role of actin in spines from different brain regions. Dendritic spines of the cerebellum have been found to contain considerably higher concentrations of actin than spines in CA1 stratum radiatum. Spines in the striatum appear to contain intermediate amounts of actin. We will model actin in spines from these three regions and use MCell to determine how the cytoskeleton affects diffusion in spines representing realistic variations in actin concentration.