title: Structures and Short Linear Motif of Disordered Transcription Factor Regions Provide Clues to the Interactome of the Cellular Hub Protein Radical-induced Cell Death1.
abstract: 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 Kdvalues. 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.
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark.
authors.forename: Charlotte
authors.initials: C
authors.lastname: O'Shea
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark.
authors.forename: Lasse
authors.initials: L
authors.lastname: Staby
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark.
authors.forename: Sidsel Krogh
authors.initials: SK
authors.lastname: Bendsen
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark.
authors.forename: Frederik Grønbæk
authors.initials: FG
authors.lastname: Tidemand
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark.
authors.forename: Andreas
authors.initials: A
authors.lastname: Redsted
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Willemoës
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark.
authors.forename: Birthe B
authors.initials: BB
authors.lastname: Kragelund
authors.affiliation: From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, 5 Ole Maaloes Vej, Copenhagen DK-2200, Denmark kskriver@bio.ku.dk.
authors.forename: Karen
authors.initials: K
authors.lastname: Skriver
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C000597112:ANAC013 protein, Arabidopsis; D029681:Arabidopsis Proteins; C114579:DREB2A protein, Arabidopsis; D009687:Nuclear Proteins; C492758:RCD1 protein, Arabidopsis; D014157:Transcription Factors
keywords: Arabidopsis thaliana; SLiM; affinity; coupled folding and binding; interactome; intrinsically disordered protein; nuclear magnetic resonance (NMR); plant stress; protein-protein interaction; thermodynamics