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. 2020 Oct 1;11:4931. doi: 10.1038/s41467-020-18708-x

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© The Author(s) 2020

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. 2 — a MAGE-A11 directly interacts with PCF11 through its MHD. In vitro translated Myc-MAGE-A11 MHD, but not N-terminal fragment, binds to recombinant GST-PCF11. Source data are provided as a Source Data file. b Overall structure of human MAGE-A11 MHD with human PCF11 degron peptide. The alpha-helical PCF11 degron peptide binds into SBC at interface of WH-A and WH-B motifs of MAGE-A11 MHD. PCF11 peptide is shown in pink and MAGE-A11 in gray. c MAGE-A11 atoms within 5 Å of PCF11 are colored cyan to indicate the SBC region. Residues in the SBC that are mutated in this study are labeled and colored yellow. d Substrate interaction is largely achieved by hydrophobic interactions along the alpha-helical interface of PCF11 with MAGE-A11 SBC. e Myc-tagged MAGE-A11 wild-type or mutants were in vitro translated and followed by in vitro binding assay with recombinant GST-PCF11 637-702 amino acids, SDS-PAGE and immunoblotting for anti-Myc. Source data are provided as a Source Data file. f MAGE-A11 SBC is crucial for PCF11 substrate binding, but not HUWE1 E3 ligase binding, in cells. HEK293FT cells stably expressing FLAG-vector, FLAG-MAGE-A11 wild-type or mutants were subjected to pull-down with anti-FLAG followed by SDS-PAGE and immunoblotting for anti-HUWE1 and anti-PCF11. Source data are provided as a Source Data file. g TR-FRET binding assays of GST-MAGE-A11 with PCF11 peptide (FLVVVHQIRQLF*Q) containing phenylalanine (black circles), 3-methyl-phenylalanine (red triangles) or 4-cyano-phenylalanine (blue squares) at the indicated position (n = 2 biologically independent experiments with triplicates per peptide). Data are mean ± SD.