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. 2017 Sep 6;7:10681. doi: 10.1038/s41598-017-11031-4

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© The Author(s) 2017

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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Structure-guided CP525 interface mutants abolish CRMP2-tubulin binding. (A,B) Size exclusion chromatography of CRMP2 mutants (blue), GTP-γS-tubulin-dimer (green), and CRMP2-mutants with GTP-γS-tubulin-dimer (red), shown with SDS-PAGE results. (A) CP525-RA. (B) CP525-NA. (C) SEC-MALS analyses of CP525 mutants and the mixtures with GTP-γS-tubulin. (D) The molar mass determined by SEC-MALS experiments. Theoretical molecular weights calculated from the amino acid sequence were also shown for comparison. (E) DLS results of CP525, CP525 mutants, GTP-γS-tubulin-dimer, and CP525/CP525 mutants with GTP-γS-tubulin-dimer. (F) Microtubule growth rate without CRMP2 (n = 7), in the presence of CP525 (n = 11), CP525-RA (n = 9), and CP525-NA (n = 9). Error bars represent the S.D. *P < 0.01; **P < 0.001. t-test. (G) Time course of microtubule polymerization after the cold-induced depolymerization without CRMP2, with CP525 wild-type or with CP525 interface mutants. A representative result from three independent experiments is shown. n = 100 cells for each transfection. *P < 0.05; **P < 0.01, compared to control. Chi-square test with Bonferroni correction. Classification is explained in Fig. 2B. (H) Percentage of end types observed. GMPCPP-microtubule (n = 187); GMPCPP-microtubule+CP525 (n = 184); GMPCPP-microtubule+CP525-RA (n = 200); GMPCPP-microtubule+CP525-NA (n = 198).