Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications Ortega-Roldán, José Luis Casares Atienza, Salvador Ringkjøbing Jensen, Malene Cárdenes, Nayra Bravo, Jerónimo Blackledge, Martin Azuaga Fortes, Ana Isabel Nuland, Nico A. J. van Binding analysis Crystal structure Crystal structure refinement Nuclear magnetic resonance Phenylalanine Protein interactions Tyrosine Ubiquitination SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. 2013-12-19T13:19:08Z 2013-12-19T13:19:08Z 2013 info:eu-repo/semantics/article Ortega Roldán, J.L.; et al. Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications. Plos One, 8(9): e73018 (2013). [http://hdl.handle.net/10481/29706] 1932-6203 doi: 10.1371/journal.pone.0073018 http://hdl.handle.net/10481/29706 eng http://creativecommons.org/licenses/by-nc-nd/3.0/ info:eu-repo/semantics/openAccess Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License Public Library of Science (PLOS)