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 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.
Highlights
Disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions
To identify the short linear motifs (SLiMs) bound by RCD1, we analyzed the Intrinsically disordered protein regions (IDRs) in three protein partners, dehydration-responsive element-binding protein 2A (DREB2A), ANAC013, and ANAC046, considering parameters such as disorder, context, charges, and pI
Because an RCD1-interacting motif has not been identified from previous studies, and because bioinformatics has come short in this endeavor, an experimental strategy is still needed for motif identification
Summary
Identification of the RCD1-binding Region in ANAC013— Discovering new functional motifs using both computational and experimental techniques is of great interest. The C-terminal ANAC013 TRD was shown to be responsible for the interaction with RCD1 (16) This 368-residue-long region is mostly disordered and contains nine short regions of predicted secondary structure, four MoRFs, and three MEME motifs (Fig. 1A). The recombinant fragment ANAC013(232–299), which showed binding to the RST domain in the yeast two-hybrid assays (Fig. 1), could be purified in amounts for analysis This truncation resulted in a significant increase in affinity with the Kd decreasing from 537 to 93 nM (Table 1; Fig. 2). The affinity increased further by additional truncation resulting in a Kd of 32 nM for ANAC013(254 –299), and removal of most of the negatively charged fragment (Fig. 1) to generate ANAC013(254 –274) resulted in a further decrease of Kd to 9.0 nM (Fig. 2A) This is a relatively low Kd value for an interaction involving a putative SLiM and a globular domain (10), and the results indicate that the ANAC013 context of the SLiM has negative allosteric effects on binding.
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