A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2

69 Pags.- 4 Figs.- Supplementary Information (6 Suppl. Tabls.- 19 Suppl. Figs.). The definitive version is available at: http://www.nature.com/nchembio/index.htmlProtein 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.We thank ARAID, MEC (BFU2010-19504, CTQ2013-­‐ 44367-­‐C2-­‐2-­‐P, CTQ2012-36365), NIH (GM061126 and CA123071) and the DGA (B89) for financial support, and BIFI (Memento cluster) for supercomputer support. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement N°283570 and BIOSTRUCTX_5186).Peer reviewe