Elucidating conformational changes in Syntaxin-1a on different stages of SNAREsassembly using Electron Paramagnetic Resonance
Bijak, Vanessa, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Cafiso, David, AS-Chemistry (CHEM), University of Virginia
When an action potential arrives at the axon terminal, it causes a local increase in intracellular Ca2+ concentration that starts a cascade of molecular interactions leading to the fusion of synaptic vesicles with the plasma membrane. This process is called neuronal exocytosis and is mediated by SNARE proteins. SNAREs are a highly conserved group of proteins that interact to form a coiled-coil assembly called the SNARE complex. The assembly of this complex is believed to provide the driving force for the fusion of the neurotransmitter-carrying vesicles to the presynaptic membrane. In the neuronal system, the SNARE complex is formed by three proteins: synaptobrevin, syntaxin-1a, and SNAP25. In addition to the SNAREs, other proteins such as Complexin, Synaptotagmin, Munc13, and Munc18 serving regulatory roles by interacting with membranes or the SNAREs.
The primary goal of this study was to investigate Syntaxin-1a, which is known to play an essential role in the process of neuronal exocytosis and signal transduction. This protein has been studied for years; however, its exact function and mechanism of action are still not fully characterized. In this study, I investigated the conformations of the plasma membrane or t-SNARE Syntaxin-1a that are believed to occur during the priming steps leading to membrane fusion. Syntaxin-1a has several domains, including a transmembrane anchor, an H3 or SNARE motif, a regulatory Habc motif, a linker that connects the H3 and Habc domains, and a short N-terminal segment called N-peptide. During the SNARE assembly process, Syntaxin-1a interacts with the SNARE chaperone Munc18. When bound to Munc18 in solution, the Habc and H3 domains are brought into proximity leading to a closed state of Syntaxin-1a. In addition to this binding mode, Munc18 also binds to the assembled SNARE complex; however, neither the mode of this interaction nor the state of the Habc regulatory domain are well characterized in this complex.
Here, I examined Syntaxin-1a using two different approaches. In the first, I used CW-EPR spectroscopy to determine if Syntaxin-1a (alone and assembled into the t-SNARE or SNARE complex) and Munc18 interact with each other. In the second, DEER spectroscopy was used to determine the conformation of Syntaxin-1a at different stages during the SNARE assembly process and to determine how Munc18 changes the conformation of Syntaxin-1a in different environments, and when it is assembled into t-SNARE or cis-SNARE complexes.
It is generally believed that when Munc18 binds to a complex of Syntaxin-1a and SNAP25, SNAP25 is dissociated, and Syntaxin-1a assumes a closed state. However, work from our laboratory indicates that Munc18 converts a 2:1 complex of Syntaxin-1a and SNAP25 into a 1:1 complex, where Syntaxin-1a is in a closed state. The work presented here supports this earlier finding and indicates in the absence of an N-terminal fragment of Syntaxin-1a, Munc18 also binds to the t-SNARE complex. Moreover, Munc18 binds to the SNARE complex through the Habc domain of Syntaxin-1a. In the case of the SNARE complex, there is no interaction between the complex and Munc18 in the absence of an N-terminal domain of Syntaxin-1a, indicating that the N-terminal domain of Syntaxin-1a is required for Munc18 to associate with the SNARE complex.
I confirmed an equilibrium between an open and closed states of Syntaxin-1a for each stage of the SNAREs assembly process. For assembled Syntaxin-1a, the equilibrium is shifted primarily towards an open state. Munc18 binds to Syntaxin-1a at each step during assembly, and shifts the equilibrium towards the closed state of Syntaxin-1a.
PHD (Doctor of Philosophy)
SNARE complex, Syntaxin-1a, Munc18, SNARE proteins, neuronal exocytosis, membrane fusion
All rights reserved (no additional license for public reuse)