Differential assembly diversifies GABAA receptor structures and signalling.

Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits1,2 and can be modulated by essential medicines including general anaesthetics and benzodiazepines3. Human GABAAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABAARs assembled from α4, ß3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors4-7. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data8,9, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABAARs.

Single-particle cryo-EM at atomic resolution.

The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the ß3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

Conformational selection guides ß-arrestin recruitment at a biased G protein-coupled receptor.

G protein-coupled receptors (GPCRs) recruit ß-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage ß-arrestins but not G proteins, making them a model system for investigating the structural basis of ß-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that ß-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular ß-arrestin-coupling region. NMR studies of ACKR3 mutants defective in ß-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides ß-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Molecular mechanism of modulating arrestin conformation by GPCR phosphorylation.

Arrestins regulate the signaling of ligand-activated, phosphorylated G-protein-coupled receptors (GPCRs). Different patterns of receptor phosphorylation (phosphorylation barcode) can modulate arrestin conformations, resulting in distinct functional outcomes (for example, desensitization, internalization, and downstream signaling). However, the mechanism of arrestin activation and how distinct receptor phosphorylation patterns could induce different conformational changes on arrestin are not fully understood. We analyzed how each arrestin amino acid contributes to its different conformational states. We identified a conserved structural motif that restricts the mobility of the arrestin finger loop in the inactive state and appears to be regulated by receptor phosphorylation. Distal and proximal receptor phosphorylation sites appear to selectively engage with distinct arrestin structural motifs (that is, micro-locks) to induce different arrestin conformations. These observations suggest a model in which different phosphorylation patterns of the GPCR C terminus can combinatorially modulate the conformation of the finger loop and other phosphorylation-sensitive structural elements to drive distinct arrestin conformation and functional outcomes.