As the prototype of Sec1/Munc18 (SM) family proteins, Munc18-1 can manipulate the distinct conformations of syntaxin-1 for controlling intracellular membrane fusion. The Munc18-1-interacting domain of Mint1 (Mint1-MID) binds to Munc18-1 together with syntaxin-1 to form a Mint1-Munc18-1-syntaxin-1 complex, but the mechanism underlying the complex assembly remains unclear. Here, we determine the structure of the Mint1-MID-Munc18-1-syntaxin-1 complex. Unexpectedly, Munc18-1 recognizes Mint1-MID and syntaxin-1 simultaneously via two opposite sites. The canonical central cavity between domains 1 and 3a of Munc18-1 embraces closed syntaxin-1, whereas the non-canonical basic pocket in domain 3b captures the acidic Mint1-MID helix. The domain 3b-mediated recognition of an acidic-helical motif is distinct from other target-recognition modes of Munc18-1. Mutations in the interface between domain 3b and Mint1-MID disrupt the assembly of the Mint1-Munc18-1-syntaxin-1 complex. This work reveals a non-canonical target-binding site in Munc18-1 domain 3b for assembling the Mint1-Munc18-1-syntaxin-1 complex.
Keywords: Mint1; Munc18-1; SM proteins; X-ray crystallography; membrane fusion.
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