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. 2020 Oct 1;11:4916. doi: 10.1038/s41467-020-18698-w

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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. 1 — a Eleven amino acid mutations of S₈-eSRK (S₈-meSRK) suppress self-aggregation. The immunoblots show the fractions from size exclusion chromatography of S₈-eSRK and S₈-meSRK ectodomains expressed in insect cells. Arrows show the elution positions of molecular mass markers. C, control supernatants of insect cell culture expressing the recombinant proteins. b S₈-meSRK possesses S₈-SP11 recognition activity. The panel shows immunoblot analysis of pull-down eluates using biotin-S₈-SP11 and insect culture media expressing S₈-meSRK or S₈-meSRK-HLH, which is an artificial fusion with the dimerization domain of a bHLH-ZIP protein¹⁸. c Chemical shift perturbation analysis of S₈-SP11. Overlay of the spectrum of ¹⁵N-labeled S₈-SP11 (blue) with that of ¹⁵N-labeled S₈-SP11 co-existing with unlabeled S₈-meSRK (red). d ITC analysis of the S₈-meSRK–S₈-SP11 interaction. Upper panel, thermogram; lower panel, integrated titration curve. e Overall structure of S₈-meSRK–S₈-SP11 heterotetramer. Two S₈-meSRK (pink and yellow) and two S₈-SP11 (cyan and green) molecules are shown as cartoon models. Dotted lines indicate disordered regions. Details of the labeled circles are shown in Fig. 2a, d, g.