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. Author manuscript; available in PMC: 2021 Apr 24.
Published in final edited form as: FEBS Lett. 2020 Dec 5;595(3):297–309. doi: 10.1002/1873-3468.14006

Figure 1. Dissecting Munc13–1 interaction with SNAP25 using Microscale Thermophoresis (MST) analysis.

Figure 1.

(A) Titration of full-length SNAP25 into AlexaFluor 660 labeled Munc13L (C1-C2B-MUN-C2C domains) produced a concentration-dependent change in the MST signal, yielding an apparent dissociation constant (Kd) = 26 ± 2 μM implying that Munc13L directly binds to SNAP25. Munc13–1 domain likely binds to the SNAP25 linker region as replacing the SNAP25 linker domain that connects that SNARE helical motifs with a GGGGS sequence (SNAP25G4S) completely abrogates Munc13-SNAP25 interaction (red curve). This interaction is highly specific as swapping the SNAP25 linker region with closely associated SNAP23 linker (SNAP25SNAP23) also disrupts the binding (blue curve).(B) MUN domain (orange curve) alone is sufficient to bind SNAP25 albeit with slightly reduced affinity (Kd = 48 ± 3 μM) and SNAP25 binding site is unique and different from the Syntaxin-1 binding site, as the Syntaxin non-binding Munc13–1 mutant (Munc13LNFAA) is capable of binding SNAP25 (purple curve). Average and standard deviations from a minimum of three independent experiments are shown.