Abstract
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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Acknowledgements
We thank S.B. Engelsen (University of Copenhagen) for providing the software to calculate 2D-RDF functions and residence times for water molecules. We thank synchrotron radiation sources DLS (Oxford) and beamline I02 (experiment number MX10121-2), and Institute for Biocomputation and Physics of Complex Systems (BIFI), (Memento cluster) for supercomputer support. This work was supported by Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Ministerio de Economía y Competitividad (MEC; BFU2010-19504 to R.H.-G., CTQ2013-44367-C2-2-P to R.H.-G., CTQ2012-36365 to F.C.), the US National Institutes of Health (GM061126 and CA123071, both to R.S.H.), the Ramón y Cajal Programme (fellowship to G.J.-O.), Diputación General de Aragón (DGA; B89 to R.H.-G.) and the EU Seventh Framework Programme (2007–2013) under BioStruct-X (grant agreement 283570 and BIOSTRUCTX 5186, to R.H.-G.).
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R.H.-G. designed the crystallization construct and solved the crystal structure. J.V.-G., E.L.-N., C.H.-R. and R.H.-G. cloned the different constructs, purified the enzymes and crystallized the complex. R.H.-G. solved and refined the crystal structure. R.H.-G. and J.V.-G. performed the ITC experiments. G.J.-O. and F.C. performed the molecular dynamics experiments. C.L.-M., D.V., H.T. and R.S.H. cloned the different constructs for expression in mammalian cells and performed site-directed mutagenesis, analysis of enzymatic studies (including the mutants in this work), study of the nonprocessivity of CePOFUT2 and studies in mammalian cells (both the secretion and the activity experiments). M.C.P. and A.L. performed the AFM studies. I.Y. and R.H.-G. performed the multiple alignment of the TSRs and POFUT2s. R.H.-G. wrote the article with contributions from H.T., R.S.H., F.C., A.L. and G.J.-O. All authors read and approved the final manuscript.
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Valero-González, J., Leonhard-Melief, C., Lira-Navarrete, E. et al. A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2. Nat Chem Biol 12, 240–246 (2016). https://doi.org/10.1038/nchembio.2019
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DOI: https://doi.org/10.1038/nchembio.2019
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