Flanking Disorder of the Folded αα-Hub Domain from Radical Induced Cell Death1 Affects Transcription Factor Binding by Ensemble Redistribution.

Protein intrinsic disorder is essential for organization of transcription regulatory interactomes. In these interactomes, the majority of transcription factors as well as their interaction partners have co-existing order and disorder. Yet, little attention has been paid to their interplay. Here, we investigate how order is affected by flanking disorder in the folded αα-hub domain RST from Radical-Induced Cell Death1 (RCD1), central in a large interactome of transcription factors. We show that the intrinsically disordered C-terminal tail of RCD1-RST shifts its conformational ensemble towards a pseudo-bound state through weak interactions with the ligand-binding pocket. An unfolded excited state is also accessible on the ms timescale independent of surrounding disordered regions, but its population is lowered by 50% in their presence. Flanking disorder additionally lowers transcription factor binding-affinity without affecting the dissociation rate constant, in accordance with similar bound-states assessed by NMR. The extensive dynamics of the RCD1-RST domain, modulated by flanking disorder, is suggestive of its adaptation to many different transcription factor ligands. The study illustrates how disordered flanking regions can tune fold and function through ensemble redistribution and is of relevance to modular proteins in general, many of which play key roles in regulation of genes.

Concerted action of the ubiquitin-fusion degradation protein 1 (Ufd1) and Sumo-targeted ubiquitin ligases (STUbLs) in the DNA-damage response.

In eukaryotes many players in the DNA-damage response (DDR) catalyze protein sumoylation or ubiquitylation. Emphasis has been placed on how these modifications orchestrate the sequential recruitment of repair factors to sites of DNA damage or stalled replication forks. Here, we shed light on a pathway in which sumoylated factors are eliminated through the coupled action of Sumo-targeted ubiquitin ligases (STUbLs) and the ubiquitin-fusion degradation protein 1 (Ufd1). Ufd1 is a subunit of the Cdc48-Ufd1-Npl4 complex implicated in the sorting of ubiquitylated substrates for degradation by the proteasome. We find that in fission yeast, Ufd1 interacts physically and functionally with the Sumo-targeted ubiquitin ligase (STUbL) Rfp1, homologous to human RNF4, and with the Sumo E3 ligase Pli1, homologous to human PIAS1. Deleting a C-terminal domain of Ufd1 that mediates the interaction of Ufd1 with Rfp1, Pli1, and Sumo (ufd1ΔCt(213-342) ) lead to an accumulation of high-molecular-weight Sumo conjugates and caused severe genomic instabilities. The spectrum of sensitivity of ufd1ΔCt(213-342) cells to genotoxins, the epistatic relationships of ufd1ΔCt(213-342) with mutations in DNA repair factors, and the localization of the repair factor Rad22 in ufd1ΔCt(213-342) cells point to ufd1ΔCt(213-342) cells accumulating aberrant structures during replication that require homologous recombination (HR) for their repair. We present evidence that HR is however often not successful in ufd1ΔCt(213-342) cells and we identify Rad22 as one of the high-molecular-weight conjugates accumulating in the ufd1ΔCt(213-342) mutant consistent with Rad22 being a STUbL/Ufd1 substrate. Suggesting a direct role of Ufd1 in the processing of Sumo-conjugates, Ufd1 formed nuclear foci colocalizing with Sumo during the DDR, and Sumo-conjugates accumulated in foci in the ufd1ΔCt(213-342) mutant. Broader functional relationships between Ufd1 and STUbLs conceivably affect numerous cellular processes beyond the DDR.