Feng Wei's research group and Xu Tao's group collaborated to reveal the regulation of membrane fusion related Mint1-Munc18-1-syntaxin...

Membrane fusion during vesicle transport is a key biological process in eukaryotic cell life activities, finely regulated
by a variety of proteins and their complexes.
The SNARE complex is the core protein molecular machine that mediates membrane fusion, assembled from SNARE
proteins.
SNARE protein is an evolutionarily conserved family of membrane-anchored proteins containing a conserved region composed of 60~70 amino acid residues, called SNARE-motif
.
When membrane fusion occurs, the four SNARE-motifs recognize and combine with each other to form a highly stable α-helical bundle structure, similar to "zipper" (SNARE-"ZIPPER"), pulling the two membranes closer together and driving the membrane fusion
.

In the nervous system, in the presynaptic membrane region, the Sec1/Munc18(SM) family proteins are key proteins
that regulate the assembly and formation of the SNARE complex.
As a member of the SM family, Munc18-1 is expressed in neurons and neuroendocrine cells, interacts directly with the SNARE protein syntaxin-1, and plays a vital role
in synaptic vesicle membrane fusion.
MUNC18-1 contains 3 domains (domains 1, 2, and 3, where domain 3 is divided into 3a and 3b) to form an arch-bridge-like structure
with a central cavity.
Among them, domain 1 binds to the N-terminal region of syntaxin-1, while the large chamber of the central region binds
to syntaxin-1 of closed conformation.
In addition, domain 3a near the central chamber has conformational flexibility, affecting interaction with SNARE
proteins.
Domain 3a of the stretched conformation can serve as an assembly template to bind to SNARE-motif of the SNARE proteins synaptobrevin-2 and syntaxin-1, facilitating the assembly
of SNARE complexes.
Mint family proteins are another key factor in the regulation of membrane fusion in the presynaptic membrane region and can interact
directly with Munc18-1.
The two main members of the Mint family of proteins (Mint1, Mint2) both contain domains that interact with Munc18-1 (MID domain) and bind to neurexin proteins on the plasma membrane to regulate plasma membrane localization
of Munc18-1.
Although it has been found that Mint1 can form complexes with Munc18-1 and syntaxin-1 and participate in the regulation of membrane fusion, the molecular mechanism of related complex assembly is unclear
.

Feng Wei's research group from the Institute of Biophysics, Chinese Academy of Sciences, and Xu Tao's research group collaborated on
this scientific problem.
Firstly, the interaction site of Mint1 and Munc18-1 was identified by biochemical means, and it was found that a disordered region rich in acidic amino acids in the MID domain of Mint1 (Mint1-MID) directly bound
to Munc18-1.
Subsequently, the structure of the Mint1-MID-Munc18-1-syntaxin-1 complex was analyzed by X-ray crystallography, and it was found that Mint1-MID and syntaxin-1 were bound to the two opposite binding sites of the Munc18-1 arch bridge-shaped structure
, respectively 。 In the complex structure, syntaxin-1 of the closed conformation binds to the central chamber enclosed by Munc18-1 domains 1 and 3a; Mint1-MID forms a short helix structure whose hydrophobic amino acids are directly inserted into the hydrophobic pocket formed by domain 3b on the back of the central chamber of Munc18-1, which is surrounded by alkaline amino acids that happen to complement the acidic amino acids in Mint1-MID with a charge
.
Mutations of key amino acids at the interface of Mint1-MID and Munc18-1 interaction, directly affect the binding of the two, disrupting the assembly of the Mint1-Munc18-1-syntaxin-1 complex
.
In summary, this study elucidates the interaction relationship between Mint1 and Munc18-1 and syntaxin-1 and the molecular mechanism of its complex assembly, revealing the mechanism
of action of Mint1 as an important scaffold protein to recognize Munc18-1 and recruit it to the plasma membrane to participate in the regulation of membrane fusion 。 The research results, titled "A non-canonical target-binding site in Munc18-1 domain 3b for assembling the Mint1-Munc18-1-syntaxin-1 complex", were published in the journal Structure on January 5, 2023
。

Illustration: Molecular mechanism by membrane fusion regulating assembly of associated Mint1-Munc18-1-syntaxin-1 complexes

Researcher Feng Wei from the Institute of Biophysics, Chinese Academy of Sciences and Song Qianli from Xu Tao's research group are the corresponding authors
of this paper.
Dr.
Li Wei of Feng Wei's research group, Xing Ying, a doctoral student in Xu Tao's group, and Wang Yue, a doctoral student in Feng Wei's group, are the co-first authors
of this paper.
The research was supported
by the National Key Research and Development Program of China, the Strategic Leading Science and Technology Project of the Chinese Academy of Sciences (B), and the National Natural Science Foundation of China.

Article link: https://doi.
org/10.
1016/j.
str.
2022.
11.
002

(Contributed by: Feng Wei Research Group)