Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Poster Abstracts

8-POS Board 8 STRUCTURAL INSIGHTS INTO MUNC13-1 SELF-ASSEMBLIES ON LIPID BILAYERS FROM CRYO-ELECTRON TOMOGRAPHY Kirill S. Grushin 1 ; Abhijith Radhakrishnan 1 ; R. Venkat Kalyana Sundaram 1 ; Jasmine S. Hinzen 2 ; Jeff Coleman 1 ; Sudhanshu Gautam 1 ; Jeremy S. Dittman 2 ; James E. Rothman 1 ; 1 Yale University, Department of Cell Biology, New Haven, CT, USA 2 Weill Cornell Medicine, Department of Biochemistry and Biophysics, New York, NY, USA Munc13-1 is a key chaperone in synaptic vesicle docking and priming, tethering vesicles to PIP2-enriched plasma membrane microdomains at the active zone and templating the SNARE proteins assembly (Syntaxin1A, VAMP2, SNAP25). Despite the critical role, the structural insights behind the functioning of Munc13 remain unclear due to conformational flexibility and the requirement for lipid membranes for its function. We recently demonstrated that the functional core of Munc13-1 (Munc13C: C1–C2B–MUN ( Δ 1408–1452, EF)–C2C, ~133 kDa) assembles into a 2D lattice between two lipid bilayers when incubated with negatively charged lipid vesicles, mimicking the synaptic active zone. Using cryo-electron tomography and subtomogram averaging we resolved the lattice at sub-nanometer resolution, revealing two membrane-bound conformations of Munc13C: an “open” form that organizes into trimers and a “closed” form that assembles into hexamers, with trimers bridging hexamers into an extended network. Mutations disrupting the oligomerization interfaces hampered vesicle docking and fusion in vitro and impaired synaptic transmission in C. elegans, underscoring the functional importance of Munc13-1 oligomerization. Furthermore, mutations targeting the hexamer supporting interface produced alternative crystal forms, revealing an additional “slanted” orientation of the open conformation, possibly reflecting a stage of Munc13-driven priming. In the slanted state, the positively charged α -helical region of the linker between C1 and C2B domains interacts with the lipid membrane surface along with C2B domain. Charge-neutralizing mutations in this region caused a dramatic reduction in vesicle docking and severely impaired Ca² ⁺ -triggered fusion in vitro. Together, these findings highlight the essential role of Munc13-1 oligomerization and conformational switching in synaptic vesicle docking and priming, supporting a stepwise model of Munc13-1 action and offering a structural framework for dissecting the molecular basis of neurotransmitter release.

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