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. 2018 May 3;8:7002. doi: 10.1038/s41598-018-25476-8

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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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(a) Superposition of E2-25K ¹H-¹⁵N HSQC NMR spectra upon titration with Ub, arrows indicate chemical shift changes with increasing Ub concentration. (b) Expansion of the spectra around G173/F174, residues experiencing significant chemical shift changes upon interaction with Ub, arrows indicate increasing Ub concentration. (c) Significant chemical shift perturbations (>Δδ + 1σ, red) upon Ub binding, mapped onto the main chain structure of E2-25K. The active site C92 (green) and gate residue Q126 (blue) are shown in the stick representation. (d) Chemical shift changes for G173 upon titration with Ub fit to a 1:1 binding isotherm, experimental shifts are shown as points, and the best fit as a surface. (e) ¹⁵N lineshape analysis for residue G173 with experimental data shown as points (blue), and the best fits shown as green lines.