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. Author manuscript; available in PMC: 2020 Oct 16.
Published in final edited form as: Cell Rep. 2020 Jul 21;32(3):107926. doi: 10.1016/j.celrep.2020.107926

Figure 2. Mapping Interactions of the CpxII Accessory Helix with the Fusion machinery by Site- and Stage-Specific Cross-linking.

Figure 2

GUVs containing the full-length t-SNARE complex (syntaxin1/SNAP25) were mixed with SUVs containing Syt1 and the v-SNARE VAMP2 in the presence of WT CpxII or CpxII BPA mutants. Samples were preincubated for 30 min on ice to accumulate docked SUVs linked to the GUVs by Syt1 and trans-SNARE complexes. UV irradiation of the reaction mix was performed before (pre-fusion) and after triggering fusion with 100 μM calcium at 365 nm for 15 s on ice. Cross-link products were analyzed by western blotting using the indicated antibodies. Colored numbers indicate CpxII BPA mutants that show prominent cross-links to SNAP25 (blue), VAMP2 (red), and synaptotagmin (gray). Positions of identified cross-link products are indicated by the molecular identity.

(A) Cross-link products identified at the prefusion stage (left panels). A view along the axis of the CpxII accessory helix shows interactions with SNAP25 (blue) and VAMP2 (red) (right panel). The membrane-proximal regions of VAMP2 and SNAP25 are depicted as cylinders because their prefusion structures are not known but may form α-helical structures.

(B) Cross-link products identified at the postfusion stage and structural model.