Neuroscience, Structural Biology
Dr. Brunger is also professor of the Department of Molecular and Cellular Physiology at Stanford University.
Molecular Mechanisms of Neurotransmitter Release
Nerve cells communicate by releasing the contents of neurotransmitter-bearing synaptic vesicles into the space between adjoining cells. This process depends on a handful of proteins that promote vesicle and nerve cell membrane fusion. The Brunger lab team uses structural and biophysical tools to capture this machinery at different stages of vesicle fusion. These structures (Figure 1) then provide the framework for further investigations, using microscopy and live neurons, into the functional and dynamic aspects of the system.
SNARE proteins, found in both nerve cell and vesicle membranes, set the stage for fusion by zipping together into a parallel, four-helix bundle that juxtaposes the two membranes. Brunger and his collaborators determined the first x-ray crystal structure of the neuronal SNARE complex, as well as the structures of other key components of the synaptic release machinery. Recently, the Brunger’s team visualized the SNARE complex bound to the Ca2+-sensor synaptotagmin-1 and to the regulator complexin, revealing two interfaces that are essential for fast synchronous release of neurotransmitters (Figure 1). The structure of this three-part complex suggests that it is in a primed and locked state. Action-potential-driven Ca2+ ions bind to the synaptotagmin proteins, unlock the complex, and trigger membrane fusion on a sub-millisecond timescale.
After fusion has occurred, SNARE complexes are recycled by the ATPase NSF, which breaks down the SNARE complex into its individual components. The Brunger team visualized this molecular machine at near-atomic level and obtained the first glimpses of how this SNARE-recycling machine works (Figure 2). The SNARE complex resembles a rope with a left-handed twist, and NSF uses adapter proteins called SNAPs to grasp the “rope” in multiple places. The SNAPs wrap around the SNARE complex with a right-handed twist, suggesting that the disassembly occurs via a simple unwinding motion that frees the zipped SNARE proteins.
The Brunger team is also using structural and functional studies to explore other machinery relevant to neurotransmitter release, such as factors involved in priming and pre-synaptic plasticity. Their research may one day provide new possibilities for targeting therapeutics to control neurotransmitter release.
A grant from the National Institutes of Health provides partial support for the work on neurotransmitter release.
Axel Brunger credits his academic success to the many disciplines he’s studied and his excellent high school courses in Germany. “It has been tremendously important to have an education in applied mathematics and physics, and then to later learn biology and biochemistry,” he says. “There are so many opportunities to apply tools and methods developed in one discipline to another.”
Early in his career, Brunger began developing tools for interpreting x-ray crystallography diffraction data. Scientists use x-ray crystallography to determine molecular structures by crystallizing the molecules and then bombarding them with x-rays. From the data produced by the diffracted x-rays, scientists can calculate a three-dimensional model of the molecule. Brunger’s powerful computational methodology revolutionized structural calculation, accelerating its automation and making protein crystallography accessible to nonexperts.
Brunger broke new ground by developing algorithms to refine crystal structures based on simulated annealing. His techniques helped identify the most accurate and lowest energy structure from x-ray crystallography diffraction data. “Simulated annealing allowed scientists to determine certain structures that before were considered intractable or required new diffraction data,” Brunger explains. He and his colleagues incorporated these algorithms into the computer program X-PLOR, as well as its successor, CNS. Their article describing CNS has been cited more than 18,000 times.
Brunger also developed a major computational tool called the “free R value,” to rate a molecular model’s quality and how likely it is to be correct. The free R value has since become a standard criterion for judging agreement between a crystallographic model and its experimental x-ray diffraction data.
Since the mid-1990s, Brunger has applied his expertise in structural biology to study the molecular mechanisms of synaptic proteins that enable nerve cell communication. At the time, scientists knew that the SNARE protein complex involved in neurotransmission consists of synaptobrevin, syntaxin-1, and SNAP-25. Synaptic vesicles carry synaptobrevin, along with neurotransmitters, to the nerve cell membrane’s inner face, which contains syntaxin-1 and SNAP-25. As the respective SNAREs zip up, they fuse the synaptic vesicle membrane and the nerve cell membrane, releasing neurotransmitter from the presynaptic neuron.
Brunger and his team showed that the corkscrew-shaped SNARE proteins assemble into quartets of one syntaxin-1, one synaptobrevin, and two SNAP-25 helices. The proteins all lie in parallel, with their heads pointing in the same direction, to promote membrane fusion.
Since moving to Stanford University in 2000, Brunger and his collaborators developed a reconstituted system that enables them to study synaptic fusion at greater level of detail than possible in neurons. The team studied the molecular mechanism of neuronal SNAREs, complexin, and synaptotagmin, as well as other factors involved in priming and pre-synaptic plasticity.
In 2015, Brunger’s team used electron cryo-microscopy to determine the structure of the supercomplex of SNAREs, the ATPase NSF, and the adapter protein α-SNAP. This subnanometer-resolution structure, along with functional studies, revealed first glimpses of the molecular mechanism of NSF-mediated SNARE complex disassembly, which allows SNARE to be recycled for the next round of synaptic vesicle fusion.
In 2017, the team determined atomic-resolution structures of the complex of the calcium sensor synaptotagmin-1, the regulator complexin, and the SNARE complex, revealing two interfaces that are essential for fast synchronous release of neurotransmitters. These structures suggest an unlocking mechanism that is triggered by Ca2+binding to the synaptotagmin molecules, leading to SNARE complex zippering, and membrane fusion.
Brunger believes that such combined structural and functional studies will eventually allow his team to elucidate the mechanisms behind Ca2+-triggered synaptic vesicle fusion.