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Molecular Mechanism of Neurotransmitter Release
Summary: Axel Brunger's goal is to understand the molecular mechanism of synaptic neurotransmission. He is particularly interested in the structure, function, and dynamics of key players in the synaptic vesicle fusion machinery. His lab is also working on the mechanism of action of clostridial neurotoxins that target this machinery. Other projects include protein complexes that are involved in synaptic development and the ATPases of the AAA family that are involved in protein complex disassembly and degradation. A molecular understanding of these complex protein machineries may ultimately lead to new therapeutics to treat human diseases.
Synaptic Vesicle Exocytosis
The pre- and postsynaptic events during neurotransmission are highly regulated and subject to stimulated changes. In the presynaptic terminal, these changes modulate the releasable pool and the release probability of synaptic vesicles. This "synaptic plasticity" could play a role in learning. The molecular components involved in neurotransmission interact in a hierarchical fashion: some components have mutual pairwise interactions, some components have interactions that are restricted to adjacent partners, and some components or groups of components are spatially separated by compartments. In addition, some of the interactions are sequential. This complexity allows the neuron to create multiple regulatory mechanisms for neuronal plasticity.
Our approach to understanding the molecular basis for neurotransmission consists of a combination of structural, functional, and dynamics studies. Structural information about the most important complexes between the individual molecular components is first obtained by x-ray crystallography, electron microscopy, or nuclear magnetic resonance spectroscopy. This information provides the framework for investigations targeted at the functional and dynamic aspects of the system, using single-molecule microscopy and spectroscopy techniques.
Among the proteins that we have intensively studied are those of the synaptic SNARE complex, consisting of syntaxin, synaptobrevin, and SNAP-25. Our crystal structure of this SNARE complex revealed an elongated, parallel four-helix bundle with a largely conserved core of interacting amino acid residues. Structures of endosomal SNARE complexes from Reinhard Jahn's group (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany), and our recent structure of the yeast SNARE complex demonstrate the conservation of these key structural features of the SNARE complex. Genetic and biochemical studies have shown that the SNARE complex plays an essential role in exocytosis. The synaptic SNARE proteins are also the exclusive targets of the protease domains of the botulinum and tetanus clostridial neurotoxins.
In Vitro Reconstitution of Ca2+-Triggered Synaptic Vesicle Fusion
The molecular mechanism that couples changes in protein-protein and protein-lipid interactions to vesicle fusion are currently unknown; proposed hypotheses are subject to considerable debate. In collaboration with Steven Chu (Lawrence Berkeley National Laboratory), we are using single-molecule fluorescence microscopy and spectroscopy to observe reconstituted SNAREs and their complexes, associated proteins, and vesicles. We monitor SNARE complex formation and docking of vesicles to deposited bilayers by illuminating the sample with laser light and observing the fluorescent signatures of dyes covalently attached at selected locations on the SNARE proteins. When fluorescent dyes are placed either on the lipids in the vesicle bilayer or alternatively in the aqueous contents of the vesicles, the spatial distribution of the fluorescent signal indicates the state of mixing of the two membranes and the degree of containment of the vesicle contents.
We have demonstrated SNARE-dependent docking of vesicles to deposited bilayers. As little as one SNARE interaction is sufficient for vesicle docking. Spontaneous fusion is a rather rare event in our assay, although the kinetics of individual fusion reactions is very fast, on the millisecond timescale. We have also studied the dynamics of the SNAREs and found that the syntaxin/SNAP-25 complex is flexible but can be stabilized by the accessory factors (in decreasing order) synaptotagmin (with and without Ca2+), complexin, Munc13, and Munc18. Ultimately, we plan to develop a model system that mimics the pertinent properties of Ca2+-triggered synaptic vesicle fusion. Such a model system should contribute to the molecular understanding of vesicle fusion since detailed biophysical measurements could be performed that are not achievable in vivo. Furthermore, such an in vitro model system could become a test bed for novel pharmaceutical drug development. (A grant from the National Institutes of Health provides partial support for this work.)
Mechanism of Action of Clostridial Neurotoxins
Clostridial neurotoxins (CNTs), such as botulinum (BoNT) and tetanus (TeNT) neurotoxins, are the causative agents of the neuroparalytic diseases tetanus and botulism. CNTs impair neuronal exocytosis by specific proteolysis of SNARE proteins once inside the neuron, resulting in the clinical manifestations of flaccid and spastic motor paralysis.
CNTs bind with high specificity at neuromuscular junctions. The molecular details of the toxin-cell recognition have been elusive. We reported the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15-Å resolution. On binding, a helix is induced in the luminal domain that binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies (collaboration with Thomas Binz and Andreas Rummel, University of Hannover, Germany) of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis.
The mechanism by which a CNT properly identifies and cleaves its target SNARE once inside the neuron involves one or more regions of enzyme-substrate interaction remote from the active site (exosites). We solved the first structure of a CNT endopeptidase in complex with its target SNARE that illustrates the extensive enzyme-substrate interface and the basis of substrate selectivity. The 2.1-Å structure of BoNT/A in complex with human SNAP-25 reveals multiple exosites, including several that are involved in substrate specificity and one that likely functions as an allosteric activator of the toxin. Together with the crystal structure of the SNARE complex, the BoNT/A complex with SNAP-25 also reveals a striking ability of the SNARE proteins to adopt very different conformations in different contexts.
The interaction between CNTs and the presynaptic release machinery is a striking example of coevolution, with the CNT receptor-binding domain choosing a protein domain for cell entry that is exposed on the surface of nerve terminals during exocytosis of synaptic vesicles and the CNT protease then cleaving synaptic SNAREs. Our results provide the basis for the development of preventive vaccines or inhibitors against these neurotoxins, as well as design of modified neurotoxins with different target specificities for clinical applications.
Synapse Development
Synapse formation and maturation are essential for the normal establishment and remodeling of neuronal circuits in the brain. Impairments in synapse formation and maturation are causes of human diseases such as autism spectrum disorders and mental retardation. Among trans-synaptic adhesion factors that play a role in synapse formation are the neurexin and neuroligin family of proteins. Neuroligins and neurexins form a complex in which two neuroligin molecules link to each other, and a neurexin molecule attaches to each side of the pair. Complex formation is downstream from cadherin and SynCAM adhesion protein engagement. As neurons create new synapses during learning, they must form neuroligin-neurexin connections for those synapses to become functionally mature.
To gain a better understanding of how the two proteins interact, my laboratory recently solved the structure of the neuroligin-1 and neurexin-1β complex. We then mutated neuroligin-1 at locations that disrupt the observed interface between it and neurexin. These mutations greatly inhibited the complex from forming, but did not affect folding of neuroligin-1. In collaboration with Thomas Südhof (HHMI, University of Texas Southwestern Medical Center at Dallas), we are now investigating how these neuroligin-1 mutations alter synaptic function, both in cultures of neurons and in mice.
Structure and Function of AAA Proteins
According to a current model, NSF (N-ethylmaleimide-sensitive factor) and SNAP (soluble NSF-attachment protein) together disassemble SNARE complexes after fusion. SNARE proteins can form both cis (same membrane) and trans (opposing membrane) complexes. It is thought that only trans-SNARE complexes lead to membrane fusion. Fusion of opposing membranes results in the formation of cis-SNARE complexes that are disassembled for recycling and reactivation by the joint action of SNAP and NSF. NSF is a hexamer and belongs to the AAA (ATPases associated with cellular activities) family of proteins. Each NSF protomer contains three domains: an N-terminal domain required for SNAP/SNARE binding and two ATPase domains, D1 and D2. ATP binding and hydrolysis by D1 are necessary for the SNARE disassembly reaction to occur, and ATP binding, but not hydrolysis, by D2 is necessary for hexamer formation. We previously solved the crystal structures of the NSF N and D2 domains.
NSF and its cofactor α-SNAP bind sequentially to SNARE complexes, forming 20S particles, named after the sedimentation behavior of the supercomplex. In collaboration with Nikolaus Grigorieff (HHMI, Brandeis University), we determined a cryo-electron microscopy structure of 20S at an estimated resolution of 11 Å. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. The crystal structure of the D2 domain of NSF was docked into the density map and shows good agreement, including at the secondary structural level. Six protrusions corresponding to the N domain of NSF emerge from the sides of the D1-domain ring.
We are also pursuing structural and functional studies of p97 (also called VCP, valosin-containing protein; VAT in archaebacteria; and cdc48 in yeast), a distant homolog of NSF. p97/VCP is involved in the degradation pathway of misfolded luminal and membrane proteins from the endoplasmic reticulum (ER). We determined the crystal structures of the biologically relevant p97/VCP hexamer in three nucleotide states: ADP; the transition-state analog of ATP hydrolysis, ADP•AlFx; and the nonhydrolyzable ATP analog, AMPPNP. The structures revealed the quarternary configuration of the individual domains and the connecting linkers, providing snapshots of p97/VCP as it proceeds through its nucleotide hydrolysis cycle. To investigate the mechanism of action of p97/VCP, we then designed a series of point mutations based on distinguishing p97/VCP structural features, such as motions during the ATP hydrolysis cycle, with particular interest in residues lining the central pore. Each mutant was tested in vitro for ATPase activity in the presence and absence of a substrate. The p97/VCP mutants were also probed in vivo for the ability to process targets for ER-associated degradation. We found that substrate dislocation is facilitated by many interactions with the D2 end of p97/VCP. Future investigations are aimed at studying the structural basis of interactions of p97/VCP with sites of retrotranslocation in the ER membrane and with ubiquitin ligases, and thus obtaining insights at the pathway of substrate translocation by p97/VCP.
Last updated: March 31, 2008
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