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In 1981, de Bold and his colleagues made the seminal observation that infusion of extracts of atrial tissue into rats caused a copious natriuresis. 1 This then led to the isolation and cloning of atrial natriuretic peptide, the first member of a family of peptides with potent natriuretic, diuretic, and vasorelaxant activity. 2 Subsequent contributions from many investigators have expanded our understanding of the family of natriuretic peptides, their receptors, and their cellular actions that regulate physiologic functions. Studies using drugs to inhibit the function of some natriuretic peptide receptors or to prevent the degradation of natriuretic peptides have confirmed the importance . . .

The MRGPRX family of receptors (MRGPRX1–4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently 1 . Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions 2 – 5 . MRGPRX2 couples to both G i and G q in mast cells 6 . Here we describe agonist-stabilized structures of MRGPRX2 coupled to G i1 and G q in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and G q . Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity. Structural studies of the itch receptors MRGPRX2 and MRGPRX4 in complex with endogenous and synthetic ligands provide a basis for the development of therapeutic compounds for pain, itch and mast cell-mediated hypersensitivity.

A protein complex composed of KPTN, ITFG2, C12orf66 and SZT2, named KICSTOR, is necessary for lysosomal localization of GATOR1, interaction of GATOR1 with the Rag GTPases and GATOR2, and nutrient-dependent mTORC1 modulation. KICSTOR is a negative regulator of mTORC1 signalling The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth and organismal homeostasis and is deregulated in many human diseases, including epilepsy and cancer. In response to nutrients, mTORC1 is recruited to the lysosome by the Rag family of GTPases, whose activity is regulated by the GATOR complex. Here David Sabatini and colleagues identify a four-membered protein complex that they term KICSTOR. It localizes to lysosomes and interacts with GATOR to negatively regulate the pathway through which mTORC1 senses nutrients. In mice lacking one of the KICSTOR subunits, SZT2, mTORC1 signalling is hyperactivated in several tissues. A related paper in this week's issue of Nature from Ming Li and colleagues identifies the protein SZT2 as a negative regulator of mTORC1 signalling. Together, the two papers offer insight into mTORC1 regulation at the lysosome and could have implications for diseases associated with hyperactive mTORC1 signalling. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy 1 , 2 , 3 . Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation 4 . Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling 5 , 6 , 7 , 8 , 9 , 10 . Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease 11 , 12 .

In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals 1 , 2 . As an MRGPR, MRGPRX2 is known to sense basic secretagogues (agents that promote secretion) and is involved in itch signals and eliciting pseudoallergic reactions 3 – 6 . MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2–G i1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2–G i1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp 6.48 to Gly 6.48 (superscript annotations as per Ballesteros–Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the G i trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions. Cryo-electron microscopy structures of the MRGPRX2–G i1 trimer in complex with polycationic compound 48/80 or inflammatory peptides provide insights into the sensing of cationic allergens by MRGPRX2, potentially facilitating the design of therapies to prevent unwanted pseudoallergic reactions.

Ketamine is a non-competitive channel blocker of N -methyl- d -aspartate (NMDA) receptors 1 . A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants 2 , 3 . Ketamine is a racemic mixture containing equal parts of ( R )- and ( S )-ketamine, with the ( S )-enantiomer having greater affinity for the NMDA receptor 4 . Here we describe the cryo-electron microscope structures of human GluN1–GluN2A and GluN1–GluN2B NMDA receptors in complex with S -ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S -ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S -ketamine moves between two distinct locations within the binding pocket. Two amino acids—leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1—were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants. Structures of ketamine bound to human NMDA receptors show how ketamine inhibits receptor activity.

Protein glycosylation is a highly important, yet poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has limited our abilities both to address the degree of heterogeneity across the glycoproteome and to understand how this contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein. Moreover, we use this large-scale glycoproteomic dataset to develop several visualizations that will prove useful for analyzing intact glycopeptides in future studies. Mass spectrometry facilitates large-scale glycosylation profiling but in-depth analysis of intact glycopeptides is still challenging. Here, the authors show that activated ion electron transfer dissociation is suitable for glycopeptide fragmentation and improves glycoproteome coverage.

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively. Two mutations affecting the sleep–wakefulness balance in mice are detected, showing that the SIK3 protein kinase is essential for determining daily wake time, and the NALCN cation channel regulates the duration of rapid eye movement sleep. Genes controlling sleep patterns Although the molecular pathways regulating circadian rhythms have been extensively explored and catalogued, much less is known about the molecular mechanisms controlling and driving sleep homeostasis. Using a forward genetic screen, Hiromasa Funato et al . identify two mutations affecting sleep/wakefulness balance. The Sik3 protein kinase was shown to be essential for determining total wake time, and mutations in the cation channel NALCN modulated REM sleep episode duration and total REM sleep time.

The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo- microscopy to determine the structure of full-length human TRPA1 to ∼4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents. The high-resolution electron cryo-microscopy structure of the full-length human TRPA1 ion channel is presented; the structure reveals a unique ankyrin repeat domain arrangement, a tetrameric coiled-coil in the centre of the channel that acts as a binding site for inositol hexakisphosphate, an outer poor domain with two pore helices, and a new drug binding site, findings that collectively provide mechanistic insight into TRPA1 regulation. Structure of multifunctional TRPA1 receptor TRP (transient receptor potential) channels are expressed by all eukaryotic organisms and act as sensors for a wide range of physical and chemical stimuli. This paper reports the high-resolution electron cryomicroscopy structure of full-length human TRPA1, a sensory receptor for noxious chemical agents such as wasabi. The overall structure of this membrane protein differs markedly from the previously published structure of TRPV1, as TRPA1 has many ankyrin repeat domains, a tetrameric coiled-coil in the center of the channel that appears to serve as a binding site for inositol hexakisphosphate and an outer pore domain with two pore helices. TRPA1 is associated with persistent pain, respiratory and chronic itch syndromes, so TRPA1 antagonists are of interest as potential analgesics.

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly ‘undruggable’ C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

N 6 -methyladenosine (m 6 A) is an abundant eukaryotic RNA modification that regulates mRNA stability. Biochemical analysis and crystallographic visualization of m 6 A-YTHDC1 interactions establish this YTH family member as an m 6 A reader and explain its RNA consensus sequence selectivity. N 6 -methyladenosine (m 6 A) is the most abundant internal modification of nearly all eukaryotic mRNAs and has recently been reported to be recognized by the YTH domain family proteins. Here we present the crystal structures of the YTH domain of YTHDC1, a member of the YTH domain family, and its complex with an m 6 A-containing RNA. Our structural studies, together with transcriptome-wide identification of YTHDC1-binding sites and biochemical experiments, not only reveal the specific mode of m 6 A-YTH binding but also explain the preferential recognition of the GG(m 6 A)C sequences by YTHDC1.