title: A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.
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.
authors: Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Zaragoza, Spain.
authors: Jessika
authors: J
authors: Valero-González
authors: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors: Christina
authors: C
authors: Leonhard-Melief
authors: Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Zaragoza, Spain.
authors: Erandi
authors: E
authors: Lira-Navarrete
authors: Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Zaragoza, Spain.|Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA.|Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, Logroño, Spain.
authors: Gonzalo
authors: http://orcid.org/0000-0003-0105-4337
authors: G
authors: Jiménez-Osés
authors: Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Zaragoza, Spain.
authors: Cristina
authors: C
authors: Hernández-Ruiz
authors: Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia de Aragón, Universidad de Zaragoza, Zaragoza, Spain.
authors: María Carmen
authors: MC
authors: Pallarés
authors: Estación Experimental de Aula Dei (EEAD-CSIC), Zaragoza, Spain.
authors: Inmaculada
authors: http://orcid.org/0000-0003-3608-4720
authors: I
authors: Yruela
authors: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors: Deepika
authors: D
authors: Vasudevan
authors: Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia de Aragón, Universidad de Zaragoza, Zaragoza, Spain.|Fundación Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Zaragoza, Spain.
authors: Anabel
authors: http://orcid.org/0000-0001-7460-5916
authors: A
authors: Lostao
authors: Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, Logroño, Spain.
authors: Francisco
authors: http://orcid.org/0000-0001-5597-8127
authors: F
authors: Corzana
authors: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors: Hideyuki
authors: H
authors: Takeuchi
authors: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors: Robert S
authors: RS
authors: Haltiwanger
authors: Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Zaragoza, Spain.|Fundación Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Zaragoza, Spain.|Instituto de Investigaciones Sanitarias de Aragón (IIS-A), Zaragoza, Spain.
authors: Ramon
authors: R
authors: Hurtado-Guerrero
mesh_terms: D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014867:Water / Q000737:chemistry*
chemical_list: D029742:Caenorhabditis elegans Proteins; D011993:Recombinant Fusion Proteins; D019700:Thrombospondin 1; D014867:Water; D005647:Fucosyltransferases; C000609579:protein O-fucosyltransferase 2, C elegans