Publications by authors named "Axel T Brunger"

139 Publications

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering.

Elife 2021 May 4;10. Epub 2021 May 4.

Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, Hanover, United States.

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.67578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143792PMC
May 2021

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies.

Neuron 2021 01 3;109(1):59-72.e5. Epub 2020 Nov 3.

Department of Pharmacology, Vanderbilt University, Nashville, TN 37240-7933, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN 37240-7933, USA. Electronic address:

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2020.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790958PMC
January 2021

Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism.

J Cell Biol 2020 09;219(9)

Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA.

Neurexins are presynaptic adhesion molecules that organize synapses by binding to diverse trans-synaptic ligands, but how neurexins are regulated is incompletely understood. Here we identify FAM19A/TAFA proteins, "orphan" cytokines, as neurexin regulators that interact with all neurexins, except for neurexin-1γ, via an unusual mechanism. Specifically, we show that FAM19A1-A4 bind to the cysteine-loop domain of neurexins by forming intermolecular disulfide bonds during transport through the secretory pathway. FAM19A-binding required both the cysteines of the cysteine-loop domain and an adjacent sequence of neurexins. Genetic deletion of neurexins suppressed FAM19A1 expression, demonstrating that FAM19As physiologically interact with neurexins. In hippocampal cultures, expression of exogenous FAM19A1 decreased neurexin O-glycosylation and suppressed its heparan sulfate modification, suggesting that FAM19As regulate the post-translational modification of neurexins. Given the selective expression of FAM19As in specific subtypes of neurons and their activity-dependent regulation, these results suggest that FAM19As serve as cell type-specific regulators of neurexin modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.202004164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480106PMC
September 2020

Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing.

EMBO J 2019 11 30;38(22):e101603. Epub 2019 Sep 30.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.

Neurexins are presynaptic, cell-adhesion molecules that specify the functional properties of synapses via interactions with trans-synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing-dependent neurexin regulation are largely unknown. Here, we determined high-resolution structures of the complex of neurexophilin-1 and the second laminin/neurexin/sex-hormone-binding globulin domain (LNS2) of neurexin-1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin-neurexin binding interface that extends the jelly-roll β-sandwich of LNS2 of neurexin-1 into neurexophilin-1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin-1. Taken together, our data reveal an unexpected architecture of neurexophilin-neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019101603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856630PMC
November 2019

The pre-synaptic fusion machinery.

Curr Opin Struct Biol 2019 02 12;54:179-188. Epub 2019 Apr 12.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, USA; Department of Structural Biology, Stanford University, Stanford, USA; Department of Photon Science, Stanford University, Stanford, USA; Howard Hughes Medical Institute, Stanford University, Stanford, USA.

Here, we review recent insights into the neuronal presynaptic fusion machinery that releases neurotransmitter molecules into the synaptic cleft upon stimulation. The structure of the pre-fusion state of the SNARE/complexin-1/synaptotagmin-1 synaptic protein complex suggests a new model for the initiation of fast Ca-triggered membrane fusion. Functional studies have revealed roles of the essential factors Munc18 and Munc13, demonstrating that a part of their function involves the proper assembly of synaptic protein complexes. Near-atomic resolution structures of the NSF/αSNAP/SNARE complex provide first glimpses of the molecular machinery that disassembles the SNARE complex during the synaptic vesicle cycle. These structures show how this machinery captures the SNARE substrate and provide clues as to a possible processing mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sbi.2019.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939388PMC
February 2019

Resolving indexing ambiguities in X-ray free-electron laser diffraction patterns.

Acta Crystallogr D Struct Biol 2019 Feb 6;75(Pt 2):234-241. Epub 2019 Feb 6.

Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.

Processing X-ray free-electron laser (XFEL) diffraction images poses challenges, as an XFEL pulse is powerful enough to destroy or damage the diffracting volume and thereby yields only one diffraction image per volume. Moreover, the crystal is stationary during the femtosecond pulse, so reflections are generally only partially recorded. Therefore, each XFEL diffraction image must be scaled individually and, ideally, corrected for partiality prior to merging. An additional complication may arise owing to indexing ambiguities when the symmetry of the Bravais lattice is higher than that of the space group, or when the unit-cell dimensions are similar to each other. Here, an automated method is presented that diagnoses these indexing ambiguities based on the Brehm-Diederichs algorithm [Brehm & Diederichs (2014), Acta Cryst. D70, 101-109] and produces a consistent indexing choice for the large majority of diffraction images. This method was applied to an XFEL diffraction data set measured from crystals of the neuronal SNARE-complexin-1-synaptotagmin-1 complex. After correcting the indexing ambiguities, substantial improvements were observed in the merging statistics and the atomic model refinement R values. This method should be a useful addition to the arsenal of tools for the processing of XFEL diffraction data sets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798318013177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400252PMC
February 2019

Structural principles of SNARE complex recognition by the AAA+ protein NSF.

Elife 2018 09 10;7. Epub 2018 Sep 10.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States.

The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (-ethylmaleimide sensitive factor) and an adaptor protein, SNAP (soluble NSF attachment protein), disassemble the SNARE complex. We report electron-cryomicroscopy structures of the complex of NSF, αSNAP, and the full-length soluble neuronal SNARE complex (composed of syntaxin-1A, synaptobrevin-2, SNAP-25A) in the presence of ATP under non-hydrolyzing conditions at ~3.9 Å resolution. These structures reveal electrostatic interactions by which two αSNAP molecules interface with a specific surface of the SNARE complex. This interaction positions the SNAREs such that the 15 N-terminal residues of SNAP-25A are loaded into the D1 ring pore of NSF via a spiral pattern of interactions between a conserved tyrosine NSF residue and SNAP-25A backbone atoms. This loading process likely precedes ATP hydrolysis. Subsequent ATP hydrolysis then drives complete disassembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.38888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160233PMC
September 2018

NSF-mediated disassembly of on- and off-pathway SNARE complexes and inhibition by complexin.

Elife 2018 07 9;7. Epub 2018 Jul 9.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States.

SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single-molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly and reassembly of individual SNARE complexes. For ternary neuronal SNARE complexes, disassembly proceeds in a single step within 100 msec. We observed short- (<0.32 s) and long-lived (≥0.32 s) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate reassembly. Either high ionic strength or decreased αSNAP concentration reduces the disassembly rate while increasing the frequency of short-lived states. NSF is also capable of disassembling anti-parallel ternary SNARE complexes, implicating it in quality control. Finally, complexin-1 competes with αSNAP binding to the SNARE complex; addition of complexin-1 has an effect similar to that of decreasing the αSNAP concentration, possibly differentially regulating cis and trans SNARE complexes disassembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.36497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130971PMC
July 2018

Molecular Mechanisms of Fast Neurotransmitter Release.

Annu Rev Biophys 2018 05;47:469-497

Department of Molecular and Cellular Physiology, Department of Neurology and Neurological Sciences, Department of Structural Biology, Department of Photon Science, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA; email:

This review summarizes current knowledge of synaptic proteins that are central to synaptic vesicle fusion in presynaptic active zones, including SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), synaptotagmin, complexin, Munc18 (mammalian uncoordinated-18), and Munc13 (mammalian uncoordinated-13), and highlights recent insights in the cooperation of these proteins for neurotransmitter release. Structural and functional studies of the synaptic fusion machinery suggest new molecular models of synaptic vesicle priming and Ca-triggered fusion. These studies will be a stepping-stone toward answering the question of how the synaptic vesicle fusion machinery achieves such high speed and sensitivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-biophys-070816-034117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378885PMC
May 2018

Ca-Triggered Synaptic Vesicle Fusion Initiated by Release of Inhibition.

Trends Cell Biol 2018 08 26;28(8):631-645. Epub 2018 Apr 26.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA; Department of Structural Biology, Stanford University, Stanford, CA, USA; Department of Photon Science, Stanford University, Stanford, CA, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.

Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca-triggered synaptic vesicle fusion. We speculate that Ca-triggered fusion commences with the release of inhibition by Ca binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca binding to one of its C2 domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tcb.2018.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056330PMC
August 2018

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography.

Biochemistry 2017 09 31;56(36):4751-4756. Epub 2017 Aug 31.

Stanford Synchrotron Radiation Lightsource , 2575 Sand Hill Road, Menlo Park, California 94025, United States.

The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.7b00711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721673PMC
September 2017

The primed SNARE-complexin-synaptotagmin complex for neuronal exocytosis.

Nature 2017 08 16;548(7668):420-425. Epub 2017 Aug 16.

Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature23484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757840PMC
August 2017

Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18.

Neuron 2017 Aug;95(3):591-607.e10

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305, USA; Department of Structural Biology, Stanford University, Stanford, CA 94305, USA; Department of Photon Science, Stanford University, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA. Electronic address:

Munc13 catalyzes the transit of syntaxin from a closed complex with Munc18 into the ternary SNARE complex. Here we report a new function of Munc13, independent of Munc18: it promotes the proper syntaxin/synaptobrevin subconfiguration during assembly of the ternary SNARE complex. In cooperation with Munc18, Munc13 additionally ensures the proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assay with SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 and Munc18 quadruples the Ca-triggered amplitude and achieves Ca sensitivity at near-physiological concentrations. In Munc13-1/2 double-knockout neurons, expression of a constitutively open mutant of syntaxin could only minimally restore neurotransmitter release relative to Munc13-1 rescue. Together, the physiological functions of Munc13 may be related to regulation of proper SNARE complex assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2017.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747255PMC
August 2017

Morphologies of synaptic protein membrane fusion interfaces.

Proc Natl Acad Sci U S A 2017 08 24;114(34):9110-9115. Epub 2017 Jul 24.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305;

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca-triggered release.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1708492114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576828PMC
August 2017

Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis.

EMBO J 2017 03 30;36(6):816-829. Epub 2017 Jan 30.

Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan, China

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1 adopts a closed conformation when bound to Munc18-1, preventing binding to synaptobrevin-2 and SNAP-25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13-1 catalyzes the transition from the Munc18-1/syntaxin-1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the syntaxin-1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18-1/syntaxin-1/MUN complex, in which syntaxin-1 still adopts a closed conformation tightly bound to Munc18-1, whereas the syntaxin-1 linker region changes its conformation, similar to that of the LE mutant of syntaxin-1 when bound to Munc18-1. We suggest that the conformational change of the syntaxin-1 linker region induced by Munc13-1 initiates ternary SNARE complex formation in the neuronal system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201695775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350566PMC
March 2017

Structure of photosystem II and substrate binding at room temperature.

Nature 2016 12 21;540(7633):453-457. Epub 2016 Nov 21.

Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the MnCaO cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S to S), in which S is the dark-stable state and S is the last semi-stable state before O-O bond formation and O evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S), two-flash illuminated (2F; S-enriched), and ammonia-bound two-flash illuminated (2F-NH; S-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the MnCaO cluster in the S and S states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature20161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201176PMC
December 2016

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes.

Proc Natl Acad Sci U S A 2016 11 7;113(47):E7590-E7599. Epub 2016 Nov 7.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305;

In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1609917113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127347PMC
November 2016

Advances in X-ray free electron laser (XFEL) diffraction data processing applied to the crystal structure of the synaptotagmin-1 / SNARE complex.

Elife 2016 10 12;5. Epub 2016 Oct 12.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States.

X-ray free electron lasers (XFELs) reduce the effects of radiation damage on macromolecular diffraction data and thereby extend the limiting resolution. Previously, we adapted classical post-refinement techniques to XFEL diffraction data to produce accurate diffraction data sets from a limited number of diffraction images (Uervirojnangkoorn et al., 2015), and went on to use these techniques to obtain a complete data set from crystals of the synaptotagmin-1 / SNARE complex and to determine the structure at 3.5 Å resolution (Zhou et al., 2015). Here, we describe new advances in our methods and present a reprocessed XFEL data set of the synaptotagmin-1 / SNARE complex. The reprocessing produced small improvements in electron density maps and the refined atomic model. The maps also contained more information than those of a lower resolution (4.1 Å) synchrotron data set. Processing a set of simulated XFEL diffraction images revealed that our methods yield accurate data and atomic models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.18740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5094853PMC
October 2016

De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure.

Nature 2016 11 28;539(7627):43-47. Epub 2016 Sep 28.

UCLA-DOE Institute for Genomics and Proteomics, Department of Biological Chemistry, University of California, Los Angeles, California 90095-1570, USA.

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals-50 unit cells per edge, on average-has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature19825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161637PMC
November 2016

Expression of C1ql3 in Discrete Neuronal Populations Controls Efferent Synapse Numbers and Diverse Behaviors.

Neuron 2016 Sep 28;91(5):1034-1051. Epub 2016 Jul 28.

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.

C1ql3 is a secreted neuronal protein that binds to BAI3, an adhesion-class GPCR. C1ql3 is homologous to other gC1q-domain proteins that control synapse numbers, but a role for C1ql3 in regulating synapse density has not been demonstrated. We show in cultured neurons that C1ql3 expression is activity dependent and supports excitatory synapse density. Using newly generated conditional and constitutive C1ql3 knockout mice, we found that C1ql3-deficient mice exhibited fewer excitatory synapses and diverse behavioral abnormalities, including marked impairments in fear memories. Using circuit-tracing tools and conditional ablation of C1ql3 targeted to specific brain regions, we demonstrate that C1ql3-expressing neurons in the basolateral amygdala project to the medial prefrontal cortex, that these efferents contribute to fear memory behavior, and that C1ql3 is required for formation and/or maintenance of these synapses. Our results suggest that C1ql3 is a signaling protein essential for subsets of synaptic projections and the behaviors controlled by these projections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2016.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017910PMC
September 2016

N-terminal domain of complexin independently activates calcium-triggered fusion.

Proc Natl Acad Sci U S A 2016 08 21;113(32):E4698-707. Epub 2016 Jul 21.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94304; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94304; Department of Structural Biology, Stanford University, Stanford, CA 94304; Department of Photon Science, Stanford University, Stanford, CA 94304; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94304

Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1604348113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987820PMC
August 2016

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion.

EMBO J 2016 08 11;35(16):1810-21. Epub 2016 Jul 11.

Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201694071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010044PMC
August 2016

: integration optimization, triage and analysis tool for the processing of XFEL diffraction images.

J Appl Crystallogr 2016 Jun 11;49(Pt 3):1057-1064. Epub 2016 May 11.

Department of Molecular and Cellular Physiology, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Department of Neurology and Neurological Science, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Department of Structural Biology, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Department of Photon Science, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.

Serial femtosecond crystallography (SFX) uses an X-ray free-electron laser to extract diffraction data from crystals not amenable to conventional X-ray light sources owing to their small size or radiation sensitivity. However, a limitation of SFX is the high variability of the diffraction images that are obtained. As a result, it is often difficult to determine optimal indexing and integration parameters for the individual diffraction images. Presented here is a software package, called , which uses a grid-search technique to determine optimal spot-finding parameters that can in turn affect the success of indexing and the quality of integration on an image-by-image basis. Integration results can be filtered using information about the Bravais lattice and unit-cell dimensions and analyzed for unit-cell isomorphism, facilitating an improvement in subsequent data-processing steps.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1600576716006683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886991PMC
June 2016

Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex.

Elife 2016 06 2;5. Epub 2016 Jun 2.

Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, United States.

Complexin regulates spontaneous and activates Ca(2+)-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.16886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927292PMC
June 2016

Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation.

Proc Natl Acad Sci U S A 2016 Apr 28;113(16):4338-43. Epub 2016 Mar 28.

Department of Bioengineering, Stanford University, Stanford, CA 94305; Department of Applied Physics, Stanford University, Stanford, CA 94305; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305

The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1600223113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843451PMC
April 2016

Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca(2+)-calmodulin.

Acta Crystallogr D Struct Biol 2016 Jan 1;72(Pt 1):83-92. Epub 2016 Jan 1.

Department of Biochemistry and Redox Biology Center, University of Nebraska, Beadle Center, Lincoln, NE 68588, USA.

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca(2+)-CaM, but no assignment of these features has been made. In addition, Ca(2+)-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca(2+)-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca(2+)-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca(2+)-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca(2+)-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca(2+)-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798315021609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756614PMC
January 2016

High-density grids for efficient data collection from multiple crystals.

Acta Crystallogr D Struct Biol 2016 Jan 1;72(Pt 1):2-11. Epub 2016 Jan 1.

Physical Bioscences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798315020847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756618PMC
January 2016

Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA+ ATPase N-Ethylmaleimide-Sensitive Factor (NSF).

J Mol Biol 2016 05 3;428(9 Pt B):1912-26. Epub 2015 Nov 3.

Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Photon Science, and Structural Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA. Electronic address:

N-ethylmaleimide-sensitive factor (NSF), first discovered in 1988, is a key factor for eukaryotic trafficking, including protein and hormone secretion and neurotransmitter release. It is a member of the AAA+ family (ATPases associated with diverse cellular activities). NSF disassembles soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes in conjunction with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP). Structural studies of NSF and its complex with SNAREs and SNAPs (known as 20S supercomplex) started about 20years ago. Crystal structures of individual N and D2 domains of NSF and low-resolution electron microscopy structures of full-length NSF and 20S supercomplex have been reported over the years. Nevertheless, the molecular architecture of the 20S supercomplex and the molecular mechanism of NSF-mediated SNARE complex disassembly remained unclear until recently. Here we review recent atomic-resolution or near-atomic resolution structures of NSF and of the 20S supercomplex, as well as recent insights into the molecular mechanism and energy requirements of NSF. We also compare NSF with other known AAA+ family members.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2015.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854814PMC
May 2016

A high-transparency, micro-patternable chip for X-ray diffraction analysis of microcrystals under native growth conditions.

Acta Crystallogr D Biol Crystallogr 2015 Oct 26;71(Pt 10):1987-97. Epub 2015 Sep 26.

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Microcrystals present a significant impediment to the determination of macromolecular structures by X-ray diffraction methods. Although microfocus synchrotron beamlines and X-ray free-electron lasers (XFELs) can enable the collection of interpretable diffraction data from microcrystals, there is a need for efficient methods of harvesting small volumes (<2 µl) of microcrystals grown under common laboratory formats and delivering them to an X-ray beam source under native growth conditions. One approach that shows promise in overcoming the challenges intrinsic to microcrystal analysis is to pair so-called `fixed-target' sample-delivery devices with microbeam-based X-ray diffraction methods. However, to record weak diffraction patterns it is necessary to fabricate devices from X-ray-transparent materials that minimize background scattering. Presented here is the design of a new micro-diffraction device consisting of three layers fabricated from silicon nitride, photoresist and polyimide film. The chip features low X-ray scattering and X-ray absorption properties, and uses a customizable blend of hydrophobic and hydrophilic surface patterns to help localize microcrystals to defined regions. Microcrystals in their native growth conditions can be loaded into the chips with a standard pipette, allowing data collection at room temperature. Diffraction data collected from hen egg-white lysozyme microcrystals (10-15 µm) loaded into the chips yielded a complete, high-resolution (<1.6 Å) data set sufficient to determine a high-quality structure by molecular replacement. The features of the chip allow the rapid and user-friendly analysis of microcrystals grown under virtually any laboratory format at microfocus synchrotron beamlines and XFELs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004715015011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601365PMC
October 2015