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

J. Valero-González, C. Leonhard-Melief, E. Lira-Navarrete, G. Jiménez-Oses, C. Hernández-Ruiz, M. C. Pallarés, I. Yruela, A. Lostao, H. Takeuchi, F. Corzana, R. S. Haltiwanger, R. Hurtado-Guerrero. Nature Chemical Biology, 12 (4), 240-246(2016)

Protein 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.

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