Molecular Mechanism of the Synaptotagmin-SNARE Complex that is Essential for Synchronous Synaptic Neurotransmitter Release

Abstract

Synaptotagmin-1 and neuronal SNARE proteins have key roles in evoked synchronous neurotransmitter release. However, it is still unknown how they cooperate to trigger synaptic vesicle fusion at the molecular level. We determined atomic-resolution crystal structures of calcium- and magnesium-bound complexes between synaptotagmin-1 and the neuronal SNARE complex. One of these structures was determined to atomic resolution with diffraction data from an X-ray free-electron laser (XFEL), leading to accurate rotamer assignments for many side chains, including those at the interfaces between molecules. This is one of the first new crystal structures determined using XFEL diffraction data. Moreover, in contrast to the tens of thousands to millions of crystals typically used in XFEL-based crystallography experiments, we obtained a reasonably complete data set from a few hundred images captured from 72 of the 148 crystals exposed to the XFEL beam. The electron density maps obtained from the XFEL diffraction data were notably superior to those of synchrotron data sets. The structures reveal several interfaces, including a large, specific, calcium independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for calcium triggered neurotransmitter release in mouse hippocampal neuronal synapses and for calcium triggered vesicle fusion in a reconstituted single vesicle fusion system. We propose that this interface forms before calcium triggering and moves en bloc as calcium influx promotes the interactions between synaptotagmin-1 and the plasma membrane. The simultaneous membrane interactions of the calcium binding loops, of the polybasic region, and of membrane-proximal region of the SNARE complex would deform the plasma membrane. This morphological change of the plasma membrane juxtaposes the membranes closer than the critical distance (0.9 nm) to promote stalk formation, and subsequently leads to fusion pore opening.