Type A γ-aminobutyric acid receptors (GABA
A
Rs) are pentameric ligand-gated ion channels (pLGICs) and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system
1
,
2
. ...Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia
3
,
4
. Amongst the numerous assemblies theoretically possible, α1β2/3γ2 GABA
A
Rs are most prevalent in the brain
5
. The β3 subunit plays an important role in maintaining inhibitory tone and expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in a CRISPR/Cas9 derived β1-3 triple knockout
6
. To date, efforts to generate accurate structural models for heteromeric GABA
A
Rs have been hampered by the use of engineered receptors and the presence of detergents
7
–
9
. Significantly, some recent cryo-EM reconstructions report “collapsed” conformations
8
,
9
which disagree with the prototypical pLGIC, the Torpedo nicotinic acetylcholine receptor
10
,
11
, the large body of structural work on homologous homopentameric receptor variants
12
, and the logic of a ion channel architecture. To address this problem, here we present a high-resolution cryo-EM structure of the full-length human α1β3γ2L, a major synaptic GABA
A
R isoform, functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator megabody and in a desensitised conformation. Unexpectedly, each GABA
A
R pentamer harbours two phosphatidylinositol 4,5-bisphosphate (PIP2) molecules, whose head groups occupy positively-charged pockets in the intracellular juxtamembrane regions of α1-subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins were not included. This structure illustrates the molecular principles of heteromeric GABA
A
receptor organization and provides a reference framework for future mechanistic investigations of GABA-ergic signalling and pharmacology.
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.
Influenza virus RNA polymerase (FluPol), a heterotrimer composed of PB1, PB2, and PA subunits (P3 in influenza C), performs both transcription and replication of the viral RNA genome. For ...transcription, FluPol interacts with the C-terminal domain (CTD) of RNA polymerase II (Pol II), which enables FluPol to snatch capped RNA primers from nascent host RNAs. Here, we describe the co-crystal structure of influenza C virus polymerase (FluPolC) bound to a Ser5-phosphorylated CTD (pS5-CTD) peptide. The position of the CTD-binding site at the interface of PB1, P3, and the flexible PB2 C-terminal domains suggests that CTD binding stabilizes the transcription-competent conformation of FluPol. In agreement, both cap snatching and capped primer-dependent transcription initiation by FluPolC are enhanced in the presence of pS5-CTD. Mutations of amino acids in the CTD-binding site reduce viral mRNA synthesis. We propose a model for the activation of the influenza virus transcriptase through its association with pS5-CTD of Pol II.
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•Influenza C virus RNA polymerase binds the CTD of RNA polymerase II•Pol II CTD binding allows the viral polymerase to snatch capped RNA primers•Pol II CTD binding stabilizes the transcriptase conformation of the viral polymerase•Pol II CTD binding enhances viral transcription
The influenza virus RNA polymerase acts both as transcriptase and replicase. Serna Martin et al. solve the structure of the influenza C virus polymerase bound to a peptide mimicking the C-terminal domain of Pol II and demonstrate that binding to Pol II stabilizes the transcriptase conformation of the viral polymerase.
Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD ...effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1-8-9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1-8-9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1-8-9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.
Interactions between genetic and environmental risk factors take center stage in the pathology of schizophrenia. We assessed if the stressor of reduced environmental enrichment applied in adulthood ...provokes deficits in the positive, negative or cognitive symptom domains of schizophrenia in a mouse line modeling NMDA-receptor (NMDAR) hypofunction in forebrain inhibitory interneurons (
). We find that
mice, when group-housed in highly enriched cages, appear largely normal across a wide range of schizophrenia-related behavioral tests. However, they display various short-term memory deficits when exposed to minimal enrichment. This demonstrates that the interaction between risk genes causing NMDA-receptor hypofunction and environmental risk factors may negatively impact cognition later in life.
Type-A γ-aminobutyric (GABA
) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the ...most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABA
receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABA
receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABA
receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABA
receptor modulators.
Type-A γ-aminobutyric receptors (GABA
A
Rs) are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among ...the most successful drugs in clinical use and common substances of abuse. Without reliable structural data, the mechanistic basis for pharmacological modulation of GABA
A
Rs remains largely unknown. Here we report high-resolution cryoEM structures of the full-length human α1β3γ2L GABA
A
R in lipid nanodiscs, bound to the channel blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA and the classical benzodiazepines alprazolam (Xanax) and diazepam (Valium), respectively. We describe the binding modes and mechanistic impacts of these ligands, the closed and desensitised states of the GABA
A
R gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding, and the transmembrane, pore-forming, regions. This work provides a structural framework to integrate decades of physiology and pharmacology research and a rational basis for development of novel GABA
A
R modulators.
Type A γ-aminobutyric acid receptors (GABA
Rs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABA
Rs to regulate acute and ...chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABA
R construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABA
R potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABA
Rs, a process of broad relevance to pentameric ligand-gated ion channels.
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 years
. 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 β3 GABA
receptor homopentamer
. 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.
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