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Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d -Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor. Here the authors reveal the structural basis of how the nicotinic acetylcholine receptor type found on skeletal muscle and in fish electric organs desensitizes in response to agonist and how the arrow poison curare antagonizes the channel by stabilizing a desensitized state.

In Archaea and Eukaryotes, the synthesis of a universal tRNA modification, N 6 -threonyl-carbamoyl adenosine (t 6 A), is catalyzed by the KEOPS complex composed of Kae1, Bud32, Cgi121, and Pcc1. A fifth subunit, Gon7, is found only in Fungi and Metazoa. Here, we identify and characterize a fifth KEOPS subunit in Archaea. This protein, dubbed Pcc2, is a paralog of Pcc1 and is widely conserved in Archaea. Pcc1 and Pcc2 form a heterodimer in solution, and show modest sequence conservation but very high structural similarity. The five-subunit archaeal KEOPS does not form dimers but retains robust tRNA binding and t 6 A synthetic activity. Pcc2 can substitute for Pcc1 but the resulting KEOPS complex is inactive, suggesting a distinct function for the two paralogs. Comparative sequence and structure analyses point to a possible evolutionary link between archaeal Pcc2 and eukaryotic Gon7. Our work indicates that Pcc2 regulates the oligomeric state of the KEOPS complex, a feature that seems to be conserved from Archaea to Eukaryotes. Many eukaryotic and archaeal tRNAs carry a modified adenosine (t 6 A) that is synthesized by the KEOPS complex, which is composed of four subunits. A fifth subunit (Gon7) is found only in fungi and metazoa. Here the authors show that archaea also possess a fifth subunit, which is structurally and functionally similar to eukaryotic Gon7.

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors. The functional role of synaptotagmin-17 (syt-17) has remained unanswered. In this study, authors demonstrate that syt-17 exists in two distinct pools in hippocampal neurons (Golgi complex and early endosomes), where it served two completely independent functions: controlling neurite outgrowth and synaptic physiology

We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation of MPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics-based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation.

Abstract DNA gyrase is a type II topoisomerase with the unique capacity to introduce negative supercoiling in DNA. In bacteria, DNA gyrase has an essential role in the homeostatic regulation of supercoiling. While ubiquitous in bacteria, DNA gyrase was previously reported to have a patchy distribution in Archaea but its emergent function and evolutionary history in this domain of life remains elusive. In this study, we used phylogenomic approaches and an up-to date sequence dataset to establish global and archaea-specific phylogenies of DNA gyrases. The most parsimonious evolutionary scenario infers that DNA gyrase was introduced into the lineage leading to Euryarchaeal group II via a single horizontal gene transfer from a bacterial donor which we identified as an ancestor of Gracilicutes and/or Terrabacteria. The archaea-focused trees indicate that DNA gyrase spread from Euryarchaeal group II to some DPANN and Asgard lineages via rare horizontal gene transfers. The analysis of successful recent transfers suggests a requirement for syntropic or symbiotic/parasitic relationship between donor and recipient organisms. We further show that the ubiquitous archaeal Topoisomerase VI may have co-evolved with DNA gyrase to allow the division of labor in the management of topological constraints. Collectively, our study reveals the evolutionary history of DNA gyrase in Archaea and provides testable hypotheses to understand the prerequisites for successful establishment of DNA gyrase in a naive archaeon and the associated adaptations in the management of topological constraints.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition 1 , 2 , yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains 3 . The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters 4 , 5 . Synaptophysin 6 and its paralogues synaptoporin 7 and synaptogyrin 8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin. Using cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles, a V-ATPase–synaptophysin interface was found to regulate synaptic vesicle biogenesis and alter seizure susceptibility.

Galloway-Mowat syndrome (GAMOS) is a severe autosomal-recessive disease characterized by the combination of early-onset steroid-resistant nephrotic syndrome (SRNS) and microcephaly with brain anomalies. To date, mutations of WDR73 are the only known monogenic cause of GAMOS and in most affected individuals the molecular diagnosis remains elusive. We here identify recessive mutations of OSGEP, TP53RK, TPRKB, or LAGE3, encoding the 4 subunits of the KEOPS complex in 33 individuals of 30 families with GAMOS. CRISPR/Cas9 knockout in zebrafish and mice recapitulates the human phenotype of microcephaly and results in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibits cell proliferation, which human mutations fail to rescue, and knockdown of either gene activates DNA damage response signaling and induces apoptosis. OSGEP and TP53RK molecularly interact and co-localize with the actin-regulating ARP2/3 complex. Furthermore, knockdown of OSGEP and TP53RK induces defects of the actin cytoskeleton and reduces migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identify 4 novel monogenic causes of GAMOS, describe the first link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.

In Archaea and Eukaryotes, the synthesis of a universal tRNA modification, t6A, is catalyzed by the KEOPS complex composed of Kae1, Bud32, Cgi121 and Pcc1. A fifth subunit, Gon7, is found only in Fungi and Metazoa. Mutations in all five genes encoding human KEOPS subunits leads to Galloway-Mowat syndrome, a severe genetic disease causing childhood lethality. Here, we describe the discovery and biochemical characterization of the archaeal fifth KEOPS subunit. This protein, dubbed Pcc2, is a paralog of Pcc1 and is widely conserved in Archaea. Pcc1 and Pcc2 form a heterodimer in solution, show modest sequence conservation but very high structural similarity. The 5-subunit KEOPS lost its capacity to form dimers but its tRNA binding and t6A synthetic activity remained robust. Pcc2 can substitute Pcc1 but the resulting KEOPS complex is inactive suggesting a distinct function for the two paralogs. Comparative sequence and structure analyses point to a possible evolutionary link between archaeal Pcc2 and eukaryotic Gon7 proteins. Our work thus reveals that Pcc2 has evolved to regulate the oligomeric state of KEOPS complex thus adding another layer of complexity to the biosynthesis of t6A that seems to be conserved from Archaea to Eukaryotes.

Alzheimer's disease (AD), a condition characterized by cognitive deficits and progressive loss of memory, is causally linked to the short amyloid peptide A 42, which disrupts normal neurotransmission. Neurotransmitter (NT) release from synaptic vesicles (SV) requires coordinated binding of the conserved core secretory machinery comprised of the soluble NSF attachment protein receptor (vSNARE) synaptobrevin 2 (VAMP2) on the SV and the cognate tSNAREs on the plasma membrane. Synaptophysin (SYP) is the most abundant SV protein3 and the major pre-fusion binding partner of VAMP24. A major challenge in understanding the etiology and prevention of AD is determining the proteins directly targeted by A 42 and elucidating if these targets mediate disease phenotypes. Here we demonstrate that A 42 binds to SYP with picomolar affinity and disrupts the SYP/VAMP2 complex resulting in inhibition of both neurotransmitter release and synaptic plasticity. While functionally redundant paralogs of SYP have masked its critical activity in knockout studies, we now demonstrate a profound seizure susceptibility phenotype in SYP knockout mice that is recapitulated in an AD model. Our studies imply a subtle yet critical role for SYP in the synaptic vesicle cycle and the etiology of AD.

Alzheimer’s disease (AD), a condition characterized by cognitive deficits and progressive loss of memory, is causally linked to the short amyloid peptide Aβ42, which disrupts normal neurotransmission1,2. Neurotransmitter (NT) release from synaptic vesicles (SV) requires coordinated binding of the conserved core secretory machinery comprised of the soluble NSF attachment protein receptor (vSNARE) synaptobrevin 2 (VAMP2) on the SV and the cognate tSNAREs on the plasma membrane. Synaptophysin (SYP) is the most abundant SV protein3 and the major pre-fusion binding partner of VAMP24. A major challenge in understanding the etiology and prevention of AD is determining the proteins directly targeted by Aβ42 and elucidating if these targets mediate disease phenotypes. Here we demonstrate that Aβ42 binds to SYP with picomolar affinity and disrupts the SYP/VAMP2 complex resulting in inhibition of both neurotransmitter release and synaptic plasticity. While functionally redundant paralogs of SYP have masked its critical activity in knockout studies5,6, we now demonstrate a profound seizure susceptibility phenotype in SYP knockout mice that is recapitulated in an AD model mouse. Our studies imply a subtle yet critical role for SYP in the synaptic vesicle cycle and the etiology of AD.