Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2.

Autor:	Weeratunga S; Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia., Gormal RS; Clem Jones Centre for Ageing and Dementia Research, Queensland Brain Institute, The University of Queensland, Queensland, Australia., Liu M; Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia., Eldershaw D; Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia., Livingstone EK; Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia., Malapaka A; Clem Jones Centre for Ageing and Dementia Research, Queensland Brain Institute, The University of Queensland, Queensland, Australia., Wallis TP; Clem Jones Centre for Ageing and Dementia Research, Queensland Brain Institute, The University of Queensland, Queensland, Australia., Bademosi AT; Clem Jones Centre for Ageing and Dementia Research, Queensland Brain Institute, The University of Queensland, Queensland, Australia., Jiang A; Clem Jones Centre for Ageing and Dementia Research, Queensland Brain Institute, The University of Queensland, Queensland, Australia., Healy MD; Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia., Meunier FA; Clem Jones Centre for Ageing and Dementia Research, Queensland Brain Institute, The University of Queensland, Queensland, Australia; School of Biomedical Sciences, The University of Queensland, Queensland, Australia., Collins BM; Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia. Electronic address: b.collins@imb.uq.edu.au.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2024 Jan; Vol. 300 (1), pp. 105541. Date of Electronic Publication: 2023 Dec 09.
DOI:	10.1016/j.jbc.2023.105541
Abstrakt:	Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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