TY - JOUR
T1 - Re-examining how Munc13-1 facilitates opening of syntaxin-1
AU - Magdziarek, Magdalena
AU - Bolembach, Agnieszka A.
AU - Stepien, Karolina P.
AU - Quade, Bradley
AU - Liu, Xiaoxia
AU - Rizo, Josep
N1 - Funding Information:
We thank Shih-Chia Tso for assistance in acquiring the ITC data and William Wickner for kindly providing purified GST-PX protein. The Agilent DD2 console of the 800 MHz spectrometer used for the research presented here was purchased with a shared instrumentation grant from the NIH (S10OD018027 to JR). Bradley Quade was supported by NIH Training Grant T32 GM008297. This work was supported by grant I-1304 from the Welch Foundation (to JR) and by NIH Research Project Award R35 NS097333 (to JR).
Publisher Copyright:
© 2020 The Protein Society
PY - 2020/6/1
Y1 - 2020/6/1
N2 - Munc13-1 is crucial for neurotransmitter release and, together with Munc18-1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin-1, SNAP-25, and synaptobrevin. Assembly starts with syntaxin-1 folded into a self-inhibited closed conformation that binds to Munc18-1. Munc13-1 is believed to catalyze the opening of syntaxin-1 to facilitate SNARE complex formation. However, different types of Munc13-1-syntaxin-1 interactions have been reported to underlie this activity, and the critical nature of Munc13-1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13-1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin-1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13-1 fragments, even though binding of this linker region to Munc13-1 is barely detectable. Conversely, the syntaxin-1 SNARE motif clearly binds to Munc13-1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13-1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13-1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin-1 via interactions with the linker.
AB - Munc13-1 is crucial for neurotransmitter release and, together with Munc18-1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin-1, SNAP-25, and synaptobrevin. Assembly starts with syntaxin-1 folded into a self-inhibited closed conformation that binds to Munc18-1. Munc13-1 is believed to catalyze the opening of syntaxin-1 to facilitate SNARE complex formation. However, different types of Munc13-1-syntaxin-1 interactions have been reported to underlie this activity, and the critical nature of Munc13-1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13-1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin-1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13-1 fragments, even though binding of this linker region to Munc13-1 is barely detectable. Conversely, the syntaxin-1 SNARE motif clearly binds to Munc13-1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13-1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13-1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin-1 via interactions with the linker.
KW - Munc13
KW - Munc18
KW - SNAREs
KW - conformational transition
KW - membrane fusion
KW - neurotransmitter release
KW - syntaxin-1
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U2 - 10.1002/pro.3844
DO - 10.1002/pro.3844
M3 - Article
C2 - 32086964
AN - SCOPUS:85081617214
SN - 0961-8368
VL - 29
SP - 1440
EP - 1458
JO - Protein Science
JF - Protein Science
IS - 6
ER -