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. 2018 Dec 11;4:67. doi: 10.1038/s41421-018-0064-8

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic representation of CRL4 ubiquitin ligase complex. The three DDB1 domains are colored and labeled BPA, BPB, and BPC. b DDA1 truncation constructs used to test interactions with DDB1. c GST pull-down assays showing direct interactions between DDB1 and DDA1 with a minimal requirement of 1–28 aa N-terminal region (NT). B beads with GST-fused bait, E elution, FL full length. d Sequence alignment and conservation of DDA1-NT across animal and plant species. e Bio-layer interferometry analysis of DDB1 binding by full-length (FL) DDA1, DDA1-NT (1–28 aa), and DDA1-CT (28–102 aa). Affinity-tagged DDA1 FL and fragments were immobilized on the probe as ligand. Free DDB1 was included as analyte at different concentration. Kd is calculated based on the dose response curve made with maximal binding signals. f Orthogonal views of the DDB1–DDA1 complex. DDA1 is shown in sticks (orange) and semi-transparent surface representation. The BPA, BPB, BPC, and CTD domain of DDB1 are shown in cyan, green, slate, and magenta cartoon diagrams. g–h Overall and close-up views of the interface between DDA1 (orange) and DDB1 BPA domain (cyan). DDA1 residues involved in DDB1 binding are shown in orange sticks. The seven blades of DDB1-BPA are labeled “1”–“7”. The four b-strands of blade “1”, “6”, and “7” are labeled “a” to “d”. i GST pull-down assay testing the binding of purified DDB1 to glutathione-beads-immobilized GST-tagged DDA1 FL, NT, and NT mutants as indicated. B, FT, and E stand for beads with GST-fusion bait, flow-through, and eluates, respectively. j In vitro reconstituted ubiquitination of GST-DDA1 (FL or NT) in the presence of wild type or lysine-less ubiquitin (denoted Ub and LLUb, respectively). k Two models illustrating the putative mode of action of DDA1 in CRL4 ligase function. DDA1 is anchored at the back side of the DDB1 BPA domain and might use its flexible C-terminal region to recruit a DCAF–substrate complex (left) and/or modulate the overall architecture of the substrate-loaded CRL4 E3 complex (right). A question mark and a ubiquitin molecule (Ub) indicate the possibility that DDA1 ubiquitination might be involved in its mode of action