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. 2020 Jun 23;11:3183. doi: 10.1038/s41467-020-16920-3

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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. 5 — a Design of a non-canonical library used in selections against 14-3-3γ. A phosphoserine was fixed in the middle, flanked by four varied positions on either side. A suite of non-canonical amino acids was incorporated at each varied position. b Sequences of putative 14-3-3γ-binding peptides (14-3-3.1, 14-3-3.6, and 14-3-3.12) identified from affinity selections, as well as a negative control peptide (non-binder or NB.1, identified as an artifact in selections against 12ca5) chosen for resynthesis and binding validation studies. Residues comprising the conserved C-term motif among 14-3-3γ binders—Nph-β-Ser-(β-Ala/β-Ser)-Nph—are indicated in blue. c Identified binders exhibited nanomolar affinities for 14-3-3γ, as determined by fluorescence anisotropy of FITC-labeled 14-3-3γ-binding peptides, NB.1, and a known 14-3-3γ-binding peptide (BiExoS; positive control). These affinities were approximately 10,000-fold higher relative to the negative control. K_D values are given in b. Uncertainties correspond to 95% confidence intervals derived from nonlinear regression. d Identified 14-3-3γ-binders could compete off bound BiExoS in a fluorescence anisotropy competition assay, suggesting they bind 14-3-3γ in the canonical, amphipathic binding groove. IC₅₀ values are given in b. e Molecular structure of 14-3-3σΔc (white surface) in complex with 14-3-3.12 (cyan sticks), based on a 1.80 Å crystal structure. The 2Fo-Fc electron density map corresponding to 14-3-3.12 is shown (blue mesh), contoured at 1σ. Abbreviations: β-Ala β-alanine; β-Ser β-homoserine; β-Thr β-homothreonine; Aad aminoadipic acid; Aph 4-aminophenylalanine; Cha cyclohexylalanine; Cpa cyclopropylalanine; Dba diaminobutyric acid; Fph 4-fluorophenylalanine; Hyp hydroxyproline; Nph 4-nitrophenylalanine; Nva norvaline; Orn ornithine; pSer phosphoserine; Thz thiazolylalanine. Error bars correspond to standard error among three technical replicates.