Structures and Short Linear Motif of Disordered Transcription Factor Regions Provide Clues to the Interactome of the Cellular Hub Protein Radical-induced Cell Death1

Intrinsically disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions. Some interactions between IDRs and folded protein domains rely on short linear motifs (SLiMs). These motifs are challenging to identify, but once found they can...

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Published in:	The Journal of biological chemistry Vol. 292; no. 2; pp. 512 - 527
Main Authors:	O'Shea, Charlotte, Staby, Lasse, Bendsen, Sidsel Krogh, Tidemand, Frederik Grønbæk, Redsted, Andreas, Willemoës, Martin, Kragelund, Birthe B., Skriver, Karen
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 13-01-2017 American Society for Biochemistry and Molecular Biology
Subjects:	affinity Amino Acid Motifs Arabidopsis - chemistry Arabidopsis - genetics Arabidopsis - metabolism Arabidopsis Proteins - chemistry Arabidopsis Proteins - genetics Arabidopsis Proteins - metabolism Arabidopsis thaliana coupled folding and binding interactome intrinsically disordered protein Models, Molecular nuclear magnetic resonance (NMR) Nuclear Magnetic Resonance, Biomolecular Nuclear Proteins - chemistry Nuclear Proteins - genetics Nuclear Proteins - metabolism Plant Biology plant stress Protein Binding Protein Folding protein-protein interaction SLiM thermodynamics Transcription Factors - chemistry Transcription Factors - genetics Transcription Factors - metabolism Arabidopsis thaliana intrinsically disordered protein coupled folding and binding nuclear magnetic resonance (NMR) thermodynamics protein-protein interaction SLiM interactome plant stress affinity
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Summary:	Intrinsically disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions. Some interactions between IDRs and folded protein domains rely on short linear motifs (SLiMs). These motifs are challenging to identify, but once found they can point to larger networks of interactions, such as with proteins that serve as hubs for essential cellular functions. The stress-associated plant protein radical-induced cell death1 (RCD1) is one such hub, interacting with many transcription factors via their flexible IDRs. To identify the SLiM bound by RCD1, we analyzed the IDRs in three protein partners, DREB2A (dehydration-responsive element-binding protein 2A), ANAC013, and ANAC046, considering parameters such as disorder, context, charges, and pI. Using a combined bioinformatics and experimental approach, we have identified the bipartite RCD1-binding SLiM as (DE)X(1,2)(YF)X(1,4)(DE)L, with essential contributions from conserved aromatic, acidic, and leucine residues. Detailed thermodynamic analysis revealed both favorable and unfavorable contributions from the IDRs surrounding the SLiM to the interactions with RCD1, and the SLiM affinities ranged from low nanomolar to 50 times higher Kd values. Specifically, although the SLiM was surrounded by IDRs, individual intrinsic α-helix propensities varied as shown by CD spectroscopy. NMR spectroscopy further demonstrated that DREB2A underwent coupled folding and binding with α-helix formation upon interaction with RCD1, whereas peptides from ANAC013 and ANAC046 formed different structures or were fuzzy in the complexes. These findings allow us to present a model of the stress-associated RCD1-transcription factor interactome and to contribute to the emerging understanding of the interactions between folded hubs and their intrinsically disordered partners.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by Norma Allewell Both authors contributed equally to this work.
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.M116.753426