doi:10.1038/nchembio.2019engValero-González, J.Leonhard-Melief, C.Lira-Navarrete, E.Jiménez-Osés, G.Hernández-Ruiz, C.Pallarés, M.C.Yruela, I.Vasudevan, D.Lostao, A.Corzana, F.Takeuchi, H.Haltiwanger, R.S.Hurtado-Guerrero, R.A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2ART-2016-94764Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.2016http://zaguan.unizar.es/record/6070810.1038/nchembio.2019http://zaguan.unizar.es/record/60708oai:zaguan.unizar.es:60708info:eu-repo/grantAgreement/ES/MINECO/CTQ2012-36365info:eu-repo/grantAgreement/ES/MICINN/CTQ2013-44367-C2-2-Pinfo:eu-repo/grantAgreement/ES/MICINN/BFU2010-19504info:eu-repo/grantAgreement/EC/FP7/283570/EU/Transnational access and enhancement of integrated Biological Structure determination at synchrotron X-ray radiation facilities/BIOSTRUCT-Xinfo:eu-repo/grantAgreement/ES/DGA/B89Nature Chemical Biology 12, 4 (2016), 240-246All rights reservedhttp://www.europeana.eu/rights/rr-f/info:eu-repo/semantics/openAccess