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. 2022 Jan 13;18(4):422–431. doi: 10.1038/s41589-021-00952-x

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© The Author(s) 2022

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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Extended Data Fig. 4 — (a) 2Fo-Fc map (1σ) is shown at the UBE2K-Ub^A binding interface. (b) Alignment of our Ub^A with known structures of Ub shows a role of Y59 in selecting a conformation of R54 at the UBE2K/Ub^A interface. (c-e) SPR analysis of UBE2K and Ub binding. Sensorgrams (left) and binding curves (right) for mono-Ub and (c) GST-UBE2K^WT, (d) GST-UBE2K^T88D and (e) GST-UBE2K^M172D,L198R. n = 2 for each binding curve with K_d indicated where applicable. Residue specific CSPs for 200 μM ¹⁵N-Ub₁ titrated until 2x molar excess of (f) UBE2K^WT and (g) UBE2K^T88D, and 5x molar excess of (h) UBE2K^M172D,L198R. No measurable binding is observed for UBE2K^M172D,L198R in either SPR or NMR. T88 is located at the UBE2K/Ub^A binding interface and UBE2K^T88D is defective in Ub^A catalysis (Fig. 3e), yet UBE2K^WT and UBE2K^T88D have near identical affinity for Ub. These data suggest that the canonical M172/L198 Ub binding surface contributes to the bulk of Ub binding affinity detected and that the UBE2K acceptor site has weak affinity for Ub^A.