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

Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase

Mutations in rumi result in a temperature-sensitive loss of Notch signaling in Drosophila. Drosophila Rumi is a soluble, endoplasmic reticulum-retained protein with a CAP10 domain that functions as a protein O-glucosyltransferase. In human and mouse genomes, three potential Rumi homologues exist: one with a high degree of identity to Drosophila Rumi (52%), and two others with lower degrees of identity but including a CAP10 domain (KDELC1 and KDELC2). Here we show that both mouse and human Rumi, but not KDELC1 or KDELC2, catalyze transfer of glucose from UDP-glucose to an EGF repeat from human factor VII. Similarly, human Rumi, but not KDELC1 or KDELC2, rescues the Notch phenotypes in Drosophila rumi clones. During characterization of the Rumi enzymes, we noted that, in addition to protein O-glucosyltransferase activity, both mammalian and Drosophila Rumi also showed significant protein O-xylosyltransferase activity. Rumi transfers Xyl or glucose to serine 52 in the O-glucose consensus sequence () of factor VII EGF repeat. Surprisingly, the second serine (S53) facilitates transfer of Xyl, but not glucose, to the EGF repeat by Rumi. EGF16 of mouse Notch2, which has a diserine motif in the consensus sequence (), is also modified with either O-Xyl or O-glucose glycans in cells. Mutation of the second serine (S590A) causes a loss of O-Xyl but not O-glucose at this site. Altogether, our data establish dual substrate specificity for the glycosyltransferase Rumi and provide evidence that amino acid sequences of the recipient EGF repeat significantly influence which donor substrate (UDP-glucose or UDP-Xyl) is used

Modulation of immune responses by targeting CD169/Siglec-1 with the glycan ligand

A fundamental role in the plant-bacterium interaction for Gram-negative phytopathogenic bacteria is played by membrane constituents, such as proteins, lipopoly- or lipooligosaccharides (LPS, LOS) and Capsule Polysaccharides (CPS). In the frame of the understanding the molecular basis of plant bacterium interaction, the Gram-negative bacterium Agrobacterium vitis was selected in this study. It is a phytopathogenic member of the Rhizobiaceae family and it induces the crown gall disease selectively on grapevines (Vitis vinifera). A. vitis wild type strain F2/5, and its mutant in the quorum sensing gene ΔaviR, were studied. The wild type produces biosurfactants; it is considered a model to study surface motility, and it causes necrosis on grapevine roots and HR (Hypersensitive Response) on tobacco. Conversely, the mutant does not show any surface motility and does not produce any surfactant material; additionally, it induces neither necrosis on grape, nor HR on tobacco. Therefore, the two strains were analyzed to shed some light on the QS regulation of LOS structure and the consequent variation, if any, on HR response. LOS from both strains were isolated and characterized: the two LOS structures maintained several common features and differed for few others. With regards to the common patterns, firstly: the Lipid A region was not phosphorylated at C4 of the non reducing glucosamine but glycosylated by an uronic acid (GalA) unit, secondly: a third Kdo and the rare Dha (3-deoxy-lyxo-2-heptulosaric acid) moiety was present. Importantly, the third Kdo and the Dha residues were substituted by rhamnose in a not stoichiometric fashion, giving four different oligosaccharide species. The proportions among these four species, is the key difference between the LOSs from both the two bacteria. LOS from both strains and Lipid A from wild type A. vitis are now examined for their HR potential in tobacco leaves and grapevine roots