Current Research and Scholarly Interests
My current research primarily focuses on the molecular mechanisms of synaptic neurotransmission. in 1998, my laboratory performed several pioneering structural studies: We determined the first X-ray crystal structure of the neuronal SNARE complex, setting the framework for subsequent functional and mechanistic studies. My laboratory also determined X-ray crystal structures of other key components of the synaptic release machinery, including parts of the ATPase NSF (N-ethylmaleimide sensitive factor), Sec17 (an α-SNAP homolog), synaptotagmin’s C2 domains, and Rab proteins and their effectors. Moreover, we determined the complex between the catalytic domain of botulinum neurotoxin A and its target SNARE (SNAP-25).
In 2015 and 2017, my lab determined crystal structures of the SNARE complex bound to the Ca2+-sensor synaptotagmin-1 and the regulator complexin at atomic resolution, revealing two interfaces, with at least one of the two being essential for fast synchronous release of neurotransmitters. The structures of these complexes suggest, along with functional studies in vitro and in neuronal cultures, 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. We used the structures of one of these two interfaces, the so-called primary interface, to develop an inhibitor of Ca2+-triggered exocytosis and mucin hypersecretion that could apply to controlling mucin-related diseases.
To complement our structural studies, we developed reconstituted systems with synaptic proteins and isolated synaptic vesicles to study synaptic vesicle fusion at the single-vesicle and single-molecule levels. This system revealed new insights about fusion pathways and the molecular mechanisms of synaptic vesicle priming and fusion.
After fusion, SNARE complexes are recycled by the ATPase NSF, which breaks down the SNARE complex into its components. This disassembly process is also essential for quality control for fusogenic SNARE complex formation in cooperation with Munc13 and Munc18. We determined structures of the so-called 20S complex consisting of the SNARE complex, α-SNAP, and NSF by single particle electron cryo-microscopy; this complex has provided first glimpses of the mechanism of this molecular recycling machine. 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. We recently discovered how the SNARE complex is loaded into NSF via side-loading and engagement of the N-terminal residues of one of the SNARE proteins (syntaxin or SNAP-25), mediated by conserved tyrosine residues in the pore of the NSF D1 ring.
Recently, we studied the molecular architecture of synapses by cryo-electron tomography. We showed that the presynaptic machinery leads to distinct inter-membrane proteinaceous interfaces in the resting state through 3D reconstructions of isolated synaptic vesicles bound to reconstituted acceptor liposomes and reconstructions of entire synapses of neural cultures. We thus uncovered that the neurotransmitter release machinery establishes stable prefusion inter-membrane complexes that facilitate fast fusion upon calcium triggering. We also discovered new interactions between macromolecules in situ: (1) Complexes of AMPA receptors with postsynaptic density scaffolding proteins (PSD). This work revealed that these molecules participate in a well-defined network that likely contributes to AMPAR stabilization and clustering. (2) A new interaction between the synaptic vesicle proteins synaptophysin and the V-ATPase. This work suggests that synaptophysin is involved in the biogenesis of synaptic vesicles by controlling the copy number of V-ATPases and possibly other molecules, such as synaptobrevin, that also interact with synaptophysin.
