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The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide.
The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide.
We used limited proteolysis–mass spectrometry (LiP–MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP–MS detects well-characterized PPIs, including antibody–target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson’s disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies.
We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.
Limited Proteolysis, Mass Spectrometry, Structural Proteomics, Protein–protein Interactions, Structure-Specific Interactions, Proteomics
Systematic identification of structure-specific protein–protein interactions.
PMID: 38702390, DOI: 10.1038/s44320-024-00037-6, S2CID: 256617477, Molecular Systems Biology:Epub ahead of print. 2024 May 3.
Aleš Holfeld, Dina Schuster, Fabian Sesterhenn, Alison K Gillingham, Patrick Stalder, Walther Haenseler, Inigo Barrio-Hernandez, Dhiman Ghosh, Jane Vowles, Sally A Cowley, Luise Nagel, Basavraj Khanppnavar, Tetiana Serdiuk, Pedro Beltrao, Volodymyr M Korkhov, Sean Munro, Roland Riek, Natalie de Souza and Paola Picotti
