BREAKTHROUGH REPORT

Structure of Arabidopsis thaliana FUT1 Reveals aVariant of the GT-B Class Fold and Provides Insight intoXyloglucan Fucosylation

Joana Rocha,a,b,1 Félix Cicéron,a,b,1 Daniele de Sanctis,c Mickael Lelimousin,a,b Valérie Chazalet,a,b

Olivier Lerouxel,a,b and Christelle Bretona,b,2

a Université Grenoble Alpes, 38400 Grenoble, FrancebCNRS, CERMAV, 38041 Grenoble cedex 9, Francec ESRF-The European Synchrotron, 38000 Grenoble, France

ORCID IDs: 0000-0003-2272-239X (J.R.); 0000-0001-7863-4327 (F.C.); 0000-0003-0391-8290 (D.d.S.); 0000-0002-4547-0132 (M.L.);0000-0002-7633-1792 (O.L.); 0000-0003-3697-0677 (C.B.)

The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, themajor hemicellulosic component in Arabidopsis thaliana, is biosynthesized in the Golgi apparatus by a series of glycansynthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosyntheticmachinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-basedbiomaterials. The xyloglucan-specific a2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose toterminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in itsapoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearlya member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-Bfold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures supportprevious findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with otherfucosyltransferases suggest that FUT1 uses a SN2-like reaction mechanism similar to that of protein-O-fucosyltransferase 2.Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi.

INTRODUCTION

Plant cells are surrounded by a strong wall comprising complexpolysaccharides and glycoproteins, which confer mechanicalproperties important for theshapeanddevelopmentofplantsand fortheir adaptation to environmental changes. The primary cell wallconsists mostly of cellulose, hemicelluloses, and pectins. Thesepolymers represent one of the largest source of renewable carbo-hydrate on Earth and therefore are of considerable interest for theproductionof bioenergy andbiomaterials (BurtonandFincher, 2014;Loqué et al., 2015). In addition, plant polysaccharides are importantsourcesofdietaryfiberwithapplications inhumanhealth (Koropatkinet al., 2012). Better knowledge of wall architecture and the finestructure of its constituents will undoubtedly lead to novel applica-tions. Similarly, identifying the molecular players of the biosyntheticmachinery will provide opportunities to manipulate cell wall com-ponents and improve plant performance for industrial applications.

The hemicelluloses are used in numerous industrial appli-cations (i.e., as food additives and in medicinal applications)

(Pauly et al., 2013). Among hemicelluloses, xyloglucan (XyG) isfound in various proportions in the primary cell walls of allangiosperms, but its fine structure depends on the species (Fry1989; Hsieh and Harris, 2009). XyG is the most abundanthemicellulose in the primary wall of dicots (making up to 20 to30% of the dry mass) (Scheller and Ulvskov, 2010). Thispolymer consists of ab-1,4-linkedglucanbackbone that canbesubstituted with a diverse array of glycosyl residues and withO-acetyl groups (Scheller and Ulvskov, 2010; Pauly andKeegstra, 2016). A one-letter code is used to describe the XyGside chains: e.g., G corresponds to the unbranched glucosylresidue, while X, L, and F correspond to glucosyl residuessubstitutedwith Xyl, Gal-Xyl, or Fuc-Gal-Xylmotifs, respectively(Fry et al., 1993). The glucan backbone is regularly substitutedwith a-D-xylosyl residues at the O6 position, but two differentcoremotifs have been described: (1) the XXXG-type (three xylosylresidues per four glycosyl residues) in dicots and commelinidmonocots and (2) the XXGGn core motif in Poaceae andSolanaceae plant species (Peña et al., 2008; Hsieh and Harris,2009). In XyG of the XXXG-type, xylosyl residues can besubstituted at O2 with b-D-Gal (L side chain) and the Galresidue further extended at O2 with a-L-Fuc (F side chain),thus forming the fucogalactoxyloglucan. Whether the struc-tural diversity of XyG is related to specific functions is yet to bedetermined, but XyG is thought to play an essential structuralrole in cell wall mechanics (Park and Cosgrove, 2015).

1 These authors contributed equally to this work.2 Address correspondence to breton@cermav.cnrs.fr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Christelle Breton (breton@cermav.cnrs.fr).www.plantcell.org/cgi/doi/10.1105/tpc.16.00519

The Plant Cell, Vol. 28: 2352–2364, October 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

The most important enzymes involved in XyG biosynthesisbelong to a large group of enzymes called glycosyltransferases(GTs). GTs catalyze the transfer of a sugar residue from an acti-vated donor (usually a nucleotide-sugar) to an acceptormolecule.They are classified as either inverting or retaining enzymes, de-pending on whether the anomeric configuration of the transferredsugar is inverted or retained in the final product. Based on aminoacid sequence similarities, GTs are currently classified into97 families (designated GTX, X corresponding to the familynumber). The 3D structures of nucleotide-sugar-dependent GTsare relatively conserved; only two types of folds (and variantsthereof), termed GT-A and GT-B, have been described to date.Most of the GTs involved in XyG biosynthesis in Arabidopsisthaliana have been identified (Pauly and Keegstra, 2016). Thebackbone is produced in theGolgi by one ormoremembers of thecellulose synthase-like family C (CSLC) (Cocuron et al., 2007).Side chains are added by Golgi-resident GTs, including a6-xylosyltransferases (XXT1, XXT2, and XXT5), b2-galactosyl-transferases (Mur3 and XLT2), and a2-fucosyltransferase (FUT1)(Perrin et al., 1999; Faik et al., 2002; Madson et al., 2003; Jensenet al., 2012; Cavalier and Keegstra, 2006; Culbertson et al., 2016).With the exception of CSLC proteins, which are multipasstransmembrane proteins, all other GTs involved in XyG bio-synthesis are type II membrane proteins consisting of a shortN-terminal cytoplasmic tail, a single transmembrane domainfollowed by a stem region, and a largeC-terminal catalytic domainfacing the luminal side.

In Arabidopsis, a single XyG:a2-fucosyltransferase (FUT1) wasshown to add the terminal fucose residue (Perrin et al., 1999, 2003;Faik et al., 2000). According to the sequence-based classification ofGTs (CAZy database, http://www.cazy.org/), FUT1 belongs to theinverting GT37 family. This family comprises only plant sequencesand includes10Arabidopsishomologs (denotedasFUT1toFUT10).FUT1 catalyzes the transfer of fucose from GDP-b-L-fucose to thesecond galactosyl residue in the XLLG and XXLG subunits, therebyproducing XLFG and XXFG subunits, respectively. However, theenzyme does not add fucose to the first galactosyl residue of XLLG,indicating that it has a strong acceptor specificity. Moreover, al-thoughtheshorteroligosaccharidesXXLGandXLLGcanfunctionasinhibitors, they are poor substrates for the enzyme, which stronglyprefers longer chains of XyG as acceptor (Faik et al., 2000; Cicéronet al., 2016). Previous analysis performed on pea (Pisum sativum)FUT1 indicated that it does not require divalent cations for activityand that the nucleotide-sugar and acceptor substrates associatewith the enzyme in a random order (Faik et al., 2000). A truncatedrecombinant form of Arabidopsis FUT1, deleted of its cytosolicdomain and transmembrane domain, has been successfully pro-duced in the baculovirus/insect cell system. The resulting proteinwas active andbehaved as a noncovalent dimer in solution (Cicéronet al., 2016). We report here the crystal structures of the catalyticdomainofArabidopsisFUT1, in its apoformand ina ternarycomplexwith its product GDP and the branched XLLG nonsaccharide ac-ceptor substrate. The structures showed a previously unreportedvariant of the classical GT-B folding. These data, together withmutant analysis, provide insights into the molecular mechanism bywhich this glycosyltransferase recognizes its donor and acceptorsubstrates and enhance our understanding of the XyG fucosylationprocess.Thisworkestablishes thestructureofaglycosyltransferase

involved in plant cell wall biosynthesis and provides the initialstructure for the entire GT37 family.

RESULTS AND DISCUSSION

Overall Structure of FUT1

We report here the crystal structures of a soluble form of Arabi-dopsis FUT1 (deletedof its first 68 aminoacids, thus excluding thecytosolic and transmembrane regions), in its apoform (2.1 Åresolution, referred to as apo-FUT1) and in complexwithGDPanda xylo-oligosaccharide fragment (2.2 Å resolution, referred to asFUT1:GDP:XLLG) (Supplemental Tables1and2).According to theDALI server (Holm and Rosenström, 2010), the closest structuralhomologs of FUT1 are fucosyltransferases belonging to differentGT families: the human a6-fucosyltransferase FUT8 (Ihara et al.,2007) and the Bradyrhizobium sp WZ9 a6-fucosyltransferaseNodZ (Brzezinski et al., 2007, 2012), both ofwhich aremembers oftheGT23 family; andGT65Caenorhabditis elegansPOFUT1 (Lira-Navarrete et al., 2011) and GT68 human POFUT2 (Chen et al.,2012), which are involved in protein O-fucosylation. All of theseproteins are members of the GT-B superfamily of glycosyl-transferases, which share the same global GT-B fold(Supplemental Figure 1). The canonical GT-B fold consists of twoRossmann-type a/b/a domains of similar size, designated as theN-domain and C-domain, separated by a large cleft where theactive site is located, and stabilized by two long C-terminala-helices (Breton et al., 2006). However, variants of the GT-B foldwith insertions anddeletions in theN-domain and/or the presenceof add-on domains have been described (for a recent review, seeBreton et al., 2012). The protein FUT1 exhibits several unusualfeatures in both domains that prompt us to classify this GT37fucosyltransferase as a GT-B fold variant.TheFUT1N-domain (residues130 to340) displaysacoreb-sheet

(Nb1 to Nb4) organized in a noncanonical order and flanked byhelicalelements (Na2toNa8)ononesideand loopsontheotherside(Figure 1). Two disulfide bridges (C111-C216 and C146-C171)connect the more exposed protein chain elements to the domaincore, including the stem and the Na3 helix. The C-domain (residues341 to 513) displays a central b-sheet (Cb1 to Cb6, strand 6 beingantiparallel to the rest) surrounded by a-helices (Ca1 to Ca6), re-sembling a Rossmann fold. The C-domain is followed by a longprotein segment (residues 514 to 558) containing two b-hairpins(Cb7-Cb8 and Cb9-Cb10) that lines the N-domain, and which canbe regarded as an extra C-terminal domain. This region establishesseveralpolar andhydrophobiccontactswith residuesofbothN-andC-domainsandmost importantly, twodisulfidebridges (C282-C530and C521-C548) firmly anchor this region to the rest of the protein(Figure 1). Interestingly, this region also plays an important role inacceptor recognition (seebelow). Thestructural organizationseen inFUT1 has not been described in other GT-B enzymes.We previously reported that the purified truncated FUT1 enzyme

behaves as a noncovalent homodimer in solution (Cicéron et al.,2016). This is corroborated by our analysis of the crystal contentsshowing that four molecules in the asymmetric unit associate intwo equivalent dimers designated as A-B and C-D (SupplementalFigure 2). The dimer formation implies a solvent occlusion of;1200 Å2, which corresponds to;6% solvent-accessible surface

Xyloglucan Fucosyltransferase Structure 2353

Figure 1. Overall Structure of FUT1.

(A) Representation of the FUT1 monomer structure. The stem region is shown in red, the N-domain is in blue, the C-domain is in green, and the extraC-domain is in orange. TheN-glycosylated asparagine residues anddisulfidebonds are in stick representation colored gray for carbon, red for oxygen, bluefor nitrogen, and yellow for sulfur atoms.(B) Topology diagram of the FUT1 structure, colored as in (A). The missing loop regions are represented in dashed lines. The N-glycosylated asparagineresidues are represented as red stars, and disulfide bonds as yellow lines, with the residue annotation.

2354 The Plant Cell

per monomer, as assessed with PISA (Protein Interfaces, Surfa-ces, and Assemblies; Krissinel and Henrick, 2007), and involvestheC-domains of the twoprotein chains (Figure 2A). PISAanalysisalso indicated that the dimer observed in the crystals has phys-iological relevance,withacomplex formationsignificancescoreof0.8. The dimer interface comprises several polar and hydrophobicinteractionsbetweenequivalent residues fromtheCa1,Ca3,Cb3,and Ca4 secondary structure elements of the two monomers(Figure 2B). In particular, W431 is almost completely buried ina hydrophobic cavity formed by the other protein chain in a plug-like interaction delimited by Ca1, Ca4, and Cb3, whereas H356stacks against its own symmetric residue.

The FUT1 structure shows that 10 of the 11 cysteine residuespresent in the protein construct are involved in intramolecularbonds (Figure 1). Moreover, the unique free cysteine (C246) isburied in the protein chain and thus is not available to establisha covalent linkage between FUT1monomers or other interactingproteins. Interestingly, using bimolecular fluorescence com-plementation assay and coimmunoprecipitation assays, Chouet al. (2015) showed that FUT1 can form disulfide-bonded ho-momers. The full-length sequence of FUT1 shows an extracysteine residue (C48) located in the transmembrane helix;therefore, it is possible that an intermolecular disulfide bondis formed between two FUT1 transmembrane helices. Such

Figure 2. Dimeric Functional Unit of FUT1.

(A)Solvent-accessible surface representation of the FUT1dimer. The color code formonomer A (left) is the sameas in Figure 1, and themonomerB (right) isshown in gray with the visible portion of its stem region highlighted in pink.(B)Residuescontactnetworkat thedimer interface.Residuesare instick representationcoloredbyelement ingreen (chainA)orgray (chainB) forcarbon, redfor oxygen, and blue for nitrogen atoms. Residues and secondary structure elements annotated underlined green or italic gray for monomers A and B,respectively.

Xyloglucan Fucosyltransferase Structure 2355

covalent association has been described in other Golgi GTs(reviewed in Kellokumpu et al., 2016).

The three-dimensional structure of FUT1 also reveals a flexibleprotein segment preceding the N-domain, corresponding to a por-tion of the stem region that connects the transmembrane domain(deleted in this protein construct) to the catalytic domain of FUT1.The stem region, which is solvent exposed, is perpendicular to theN- andC-domain interfacewhere the initial Na1helix stacks againsthelical elements of the C-domain (Figure 1). Electrostatic surfaceanalysisof theFUT1dimershowscontrastedviewswithaprominentpositively charged “membrane-facing” surface and a negativelycharged “luminal side,”with theexceptionof thepositiveGDP/GDP-fucose binding pocket that is located on the luminal side (Figure 3).From these observations, one can hypothesize that the FUT1 dimermay interact through itsmembrane sidewith the negatively chargedmembrane bilayer. The FUT1 protein construct used in this studyharbors two N-glycosylation sites, at positions N88 (in the stemregion) and N504 (in the C-domain), which according to previousMALDI-TOF analysis are fully occupied (Cicéron et al., 2016). Thetwo asparagine residues are solvent exposed but there is weak

electron density near each Asn residue, indicating the likely at-tachment of N-glycan chains at these sites. The comparison be-tween apo-FUT1 and FUT1:GDP:XLLG structures reveals no majorconformational changes in the polypeptide chains. The two struc-tures are almost identical and only subtle loop movements in thevicinityof thedonorandacceptorbindingpocketsareobserved.TheCb1-Ca2 loop (V365-F375) moves;2 Å toward the donor bindingsite, whereas the Cb5-Cb6 loop (L499-C512) moves slightly (;1 Å)toward the XLLG acceptor binding site, leading to rearrangement ofseveral residue side chains such as R366 or R501 (see below).

Donor Sugar Binding Site

Despite extensive attempts to obtain FUT1 in complex with itsdonor substrate (GDP-fucose), our crystals always showed clearelectron density only for the GDP moiety. This can be attributedeither to the flexibility and/or disorder of fucose residue since it isexposed to the solvent or to the hydrolysis of the donor substrate.In all structures, the GDP ligand occupies the same position as inthe higher resolution FUT1:GDP:XLLG structure (Figure 4A). Theguanine is sandwichedbetween theside chainsof F484andL418,and the carboxylate group of E466 establishes two H-bonds withthe N1 and N2 atoms of the guanine ring (Figure 4B). The riboseestablishes two hydrogen bonds, with a water molecule and withH459sidechain.Thenucleotidedonor isfirmlyheld inplacemainlyby the diphosphate moiety, which forms an intricate network ofpolar interactions with several protein residues. The a-phosphateinteracts with the backbone amides from G183 and N184, whichare theonly residues from theN-domain that interactwith theGDPmoiety. The O3A atom of the phosphate group establishes twohydrogenbondswith T483side chain andF484main chain amide.The b-phosphate establishes a total of five hydrogen bonds withS482 and T483 side chains, with R366 NE and NH1 side chainatoms and a solvent water molecule.Even though FUT1 and its structural homologs FUT8, NodZ,

POFUT1, and POFUT2 share <20% overall sequence identity,comparison of these structures revealed the same overall GDP-fucose bindingmodewith identical or similar residues in the samespatial location in their C-domains (Supplemental Figure 3). Themost conserved residues belong to three peptidemotifs (denotedas I, II, and III) that have been previously identified in fucosyl-transferases belonging to five different CAZy families (GT11,GT23, GT37, GT65, and GT68) (Chazalet et al., 2001; Martinez-Dunckeretal., 2003). The importanceof someof these residues foractivity hasbeendemonstratedbymutational analyses for severalenzymes.Ofparamount importance is the invariantarginine residue(R366 inFUT1,motif I), located at theCb1-Ca2 loop inFUT1,whichinteracts in a conserved manner with the b-phosphate of GDP. Itsmutation in alanine (or lysine) abolished enzyme activity of FUT8(Takahashi et al., 2000), NodZ (Chazalet et al., 2001), POFUT1(Lira-Navarrete et al., 2011), and POFUT2 (Chen et al., 2012), thushighlighting its crucial role in donor sugar binding. Four otheramino acids are similarly positioned in the GDP/GDP-fucosebindingpocket in all fucosyltransferase structures, correspondingtoS482, T483, F484, andE466 inFUT1. The [482-S-T-F] sequence,located at the beginning of Ca5, is part of motif III, whose functionseems to be the correct positioning of pyrophosphate and fucosemoieties for the transfer reaction. The acidic position equivalent to

Figure 3. Electrostatic Surface Representation of the FUT1 Dimer.

Views from the luminal and membrane-facing sides are displayed. Thedimer interface is indicated as a black dashed line and the colored arrowsshow the binding sites for the GDP donor (red) and XLLG acceptor (black)substrates.

2356 The Plant Cell

E466 was also shown to be critical for activity in FUT8 and NodZ(Ihara et al., 2007; Chazalet et al., 2001). The most conservedresidues in these peptide motifs are therefore key anchoringpoints for the guanine and diphosphate moieties (Supplemental

Figure 3). It is worth noting that the three peptide motifs are alsoobserved inother Arabidopsis FUTproteins of familyGT37 (Figure5A). However, the FUT3 sequence lacks the conserved arginineresidue in motif I, and FUT9 lacks motif II.

Figure 4. Structure of the FUT1:GDP:XLLG Ternary Complex.

(A)Surface representationofFUT1 (coloredas inFigure1) incomplexwithGDPandXLLG.Thebindingsitesare locatedon the luminal sideofFUT1,oppositeto the stem region.(B)Viewof theGDP ligand in the FUT1 donor binding pocket. TheGDPelectron density Fo-Fcmap at 2.2 Å is represented in greenmesh contoured at 3.0s.Ligand andprotein residues in stick representation colored yellow (GDP) or gray (protein) for carbon atoms, blue for nitrogen, red for oxygen, and orange forphosphorous. Water molecules are represented as red spheres.(C) Schematic representation of XLLG, a xylo-nonsaccharide acceptor, showing the b1,4-glucan backbone with side chains made of xylose or xylose-galactose. FUT1 transfers a fucose residue to the OH group of Gal3 (dashed red box).(D)Viewof theXLLGacceptorbindingpocket. Ligandandprotein residues instick representationarecoloredas in (B).Residuesannotated inorangeare fromtheextraC-domain. The XLLG Fo-Fc electron density map at 2.2 Å is represented in green mesh contoured at 3.0s. Water molecules are represented as red spheres.

Xyloglucan Fucosyltransferase Structure 2357

Acceptor Substrate Binding Site

The ternary complex FUT1:GDP:XLLG provides insight into theacceptor binding site of the enzyme.Weobtained this complex bysoaking apo-FUT1 crystals with both ligands. Clear electrondensity for the bound GDP was observed in each of the fourmonomers in theasymmetricunit.Bycontrast, electrondensity forthe bound XLLG molecule was visible in only two monomers(B and C). This could be attributed to crystallographic symmetryconstraints. As shown in Supplemental Figure 4, access to theXLLGbinding site inmonomers A andD is obstructed by contactswith symmetry-relatedmolecules. Therefore, it seemsplausible toconsider that both acceptor binding sites are accessible in thefunctional dimer. The oligosaccharide XLLGcanbe considered asoneof theminimal acceptor structures for FUT1. It is composed ofa b-1,4-D-glucan backbone with branches made of Xyl and Xyl-Gal. For clarity, all sugars have been labeled as indicated in Figure4C. The FUT1 enzyme transfers a fucose selectively to the O2position of Gal3 in XLLG, forming a a1-2 linkage in the resulting

product XLFG. The XLLG binding site is solvent exposed and, likethe donor binding pocket, it locates opposite to the stem region(Figure 4A). Several water-mediated and direct interactions areestablished between the XLLG ligand and FUT1 residues from theN-domain and extra C-terminal region. The oligosaccharide isheld in place mainly by contacts with Xyl1 and Gal3 moieties(Figure 4D). Although most of the acceptor monosaccharidescould be accurately modeled in the electron density maps of twoFUT1monomers, the Xyl2 andGal2moieties are solvent exposedand do not fit well in density, thus reflecting the flexibility for thispart of the ligand. As illustrated in Figure 4C, these two residuespoint in opposite directions from Xyl1 and Xyl3-Gal3. The O3 andO4 atoms of the Xyl1 moiety interact with the K278 NZ atom.Furthermore, Xyl1 O4 is also involved in H-bond formation withP525 backbone amide, while the O6 atom interacts with the sidechain of S524. TheW553 residue stacks against theGlc1 ring andalso hydrogen bonds to theO3 atomof Glc2 via awatermolecule.The R501 side chain interacts both with Xyl2 O4 and Glc3 O2atoms. This protein residue locates in the Cb5-Cb6 loop that

Figure 5. Multiple Sequence Alignment of Arabidopsis FUT Protein Sequences of Family GT37.

(A)Amino acid composition of conserved peptidemotifs I, II, and III in FUT1 to FUT10 sequences. Residues in bold red in FUT1 are involved inGDPbinding.FUT3 sequence lacks the conserved arginine variant in motif I. FUT9 lacks motif II.(B) All proteins except FUT3 contain the extra C-terminal domain with its four conserved cysteines (yellow shading). Residues in bold blue are involved inXLLG acceptor binding. FUT3 harbors a truncated extra C-domain.(C) Amino acid conservation of catalytic residues in FUT sequences.

2358 The Plant Cell

moves upon substrate binding. The Gal2, Xyl3, and Glc4 sugarresidues do not contact protein residues, directly or via solventinteractions. By contrast, all the hydroxyl groups of the Gal3moiety are involved in protein and/or solvent interactions. The C6hydroxyl establishes twohydrogenbondswithS524OGatomandwith N301 side chain amine. The C4 hydroxyl establishes threeH-bonds,with theH523sidechain, theW481mainchainamideviaa solvent molecule, and the Glc2 O2 atom, in an intramolecularwater-mediated H-bonding. Two solvent-mediated contacts areestablished between the Gal3 C3 hydroxyl group and the mainchain carbonyl of D300 and W481. Moreover, it hydrogen bondswith the N184 side chain that also interacts with the O2 atom ofGal3. Curiously, N184 tightly interacts with the S180 side chain,which isanoutlier in theRamachandranplot (S180 isalsoanoutlierin the apo-FUT1 structure).The FUT1:GDP:XLLG structure provides insight into the ac-

ceptor specificity of FUT1. The data clearly demonstrate thatFUT1 can accommodate both XLLG and XXLG oligosaccharideacceptors, as it was previously shown for the pea FUT1 enzyme(Faiketal., 2000), andexplainwhyonlyGal3 (butnotGal2) inXLLG/XXLG can be fucosylated, thus forming the XLFG and XXFGproducts. Previous studies highlighted the strong preference ofFUT1 for longer chains of XyG as acceptor (Faik et al., 2000). Onecan anticipate from our structural data that a polymeric acceptorsubstrate, which is also a multivalent ligand, would make addi-tional contacts with the FUT1 enzyme and/or that occupancy ofboth acceptor binding sites in the dimer by a single moleculewould create a cluster glycoside effect, thus resulting in a muchhigher affinity of FUT1 for theXyGpolymer (Figure3). As shown inFigure 4D, the acceptor is held in place through contactswith aminoacid residues of theN-domain andof the extraC-terminal region. Asdescribed above, the latter region, which is unique to FUT1, ischaracterizedby the presenceof twob-hairpins and four conservedcysteines that are engaged in disulfide bonds (Figure 1). In ourstructure, the sequence motif [523-H-S-P] appears to contribute tothecorrectanchoringofXLLG/XXLGXyGsubunits. Interestingly, thisextra C-terminal region is also present in the nine other ArabidopsisFUT sequences of family GT37 (Figure 5B). The extent of overallsequence identity to FUT1 polypeptide sequence ranges from;38to 55% (Sarria et al., 2001).In Arabidopsis, FUT1 is currently considered the sole fuco-

syltransferase active on XyG. The precise biochemical function ofthe other FUT proteins remains largely unknown, except for theArabidopsisFUT4andFUT6proteins,whichwereshown tobea2-fucosyltransferases acting on arabinogalactan proteins (Wu et al.,2010). Ifwehypothesize that theextraC-terminal region issimilarlyinvolved in acceptor binding in other FUT proteins, then threesubgroups can be defined on the sole basis of their corre-sponding [523-H-S-P]sequences:FUT2andFUT10are theclosest toFUT1 and can be grouped together; FUT4 to FUT7 form anothersubgroup; FUT8 and FUT9 can also be grouped, although FUT9lacks the peptide motif II (Figure 5A). FUT3 appears as the mostatypical member andmay not even be a fucosyltransferase, since itlacks the invariant catalytic arginine residue andharbors a truncatedC-terminal region. We anticipate that this grouping may help todecipher the precise biochemical function of the other plant FUTmembers. Interestingly, an Arabidopsis mutant named mur2 har-boring a point mutation in the FUT1 sequence has been previously

Figure 6. Proposed Reaction Mechanism for FUT1.

(A) The active site of FUT1 in complex with GDP-fucose and XLLG. Thefucose moiety was modeled based on the structure of the POFUT1 ho-molog. Protein residuesand ligandsaredisplayed assticks, colored red foroxygen, blue for nitrogen, orange for phosphorous, and gray, green, oryellow for amino acid, GDP-fucose, or XLLG carbon atoms, respectively.Protein-ligand interactions are depicted as black dashes.(B) FUT1 activity assays of mutants compared with wild-type enzyme(average 6 SD, n = 3).(C) Proposed reaction mechanism for FUT1. The enzyme reaction isproposed to occur through a single-displacement SN2 mechanism viaan oxocarbenium ion-like transition state assisted by a catalytic base,D300.

Xyloglucan Fucosyltransferase Structure 2359

described (Vanzin et al., 2002). The mur2 mutation, D550N, whichabolished enzyme activity as compared with the wild-type enzyme,is located in the extra C-terminal region and may thus affect theacceptor binding site. However, this mutation also creates an ad-ditionalN-glycosylationsiteandonecannotexcludethatoccupancyof this site by aN-glycanwould impair thecorrect foldingof theextraC-terminal region and consequently the acceptor binding site.

Proposed Reaction Mechanism for FUT1

Even though no FUT1:GDP-fucose structure is available at themoment, comparison of the FUT1:GDP:XLLG structure with theclosest homologsPOFUT1 (Lira-Navarrete et al., 2011) andPOFUT2(Chen et al., 2012), for which a complex with GDP-fucose isavailable, allowed modeling of the fucose residue in the bindingsite (Figure 6A). Due to the close proximity of Gal3 of the acceptorsubstrate and the pyrophosphate moiety of the GDP, and con-sidering that the fucose anomeric carbon has to locate near theGal3 C2 atom for the transfer reaction to occur, the positioning ofthe fucose ring was straightforward. The side chains of F368 andW481, together with the Glc3 and Glc4 acceptor sugar residues,create a cavity (just belowGal3) that can easily accommodate thefucose ring. The protein-ligand interactions observed for theguanosine moiety are essentially the same in both models (GDPversus GDP-fucose), but the position of the pyrophosphategroups is slightly different, and new H-bonds are established(Figure 6A; Supplemental Figure 5). In the GDP-fucosemodel, thea-phosphatenowinteractswithG183main-chainamideandT483side chain, while the b-phosphate only contacts the T483 side-chain and main-chain amide, and the R366 NE atom. The fucosemoiety establishes two hydrogen bondswith the N184 side-chainand the acceptor Xyl3 O3 atom. Interestingly, the presence of thefucose induces a movement of Gal3 moiety upwards close to theD300 side chain (Supplemental Figure 5).

FUT1 and its structural homologs are inverting glycosyl-transferases. Inverting GT reactions are suggested to occur ina single displacement SN2 mechanism via an oxocarbenium ion-like transition state assisted by a catalytic base (Lairson et al.,2008; Breton et al., 2012). An aspartate, glutamate, or histidineusually serves as a catalytic base to deprotonate the nucleophilehydroxylgroupof theacceptor substrate. InPOFUT2,aconservedglutamate residue (E54) in the N-domain is appropriately posi-tioned to be proposed as the catalytic base (Supplemental Figure6A) (Chen et al., 2012). In addition, its mutation to alanine totallyabolished enzyme activity, which is consistent with a role asageneral base.Contrary towhatwasexpected for an invertingGT,no catalytic base could be identified in the active site of POFUT1.Instead, mutagenesis data pointed to the catalytic importance oftwo amino acid residues (R240 and N43 in POFUT1), and a SN1-like mechanism has been proposed for this protein-O-fucosyl-transferase 1 (Supplemental Figure 6B) (Lira-Navarrete et al.,2011). In this model, the cleavage of the glycosidic bond wouldtake place first, leading to the formation of an intimate ion pair inthe transition state. A similar mechanism, intermediary betweenSN1 and SN2 reaction, has been proposed for the human a3-fucosyltransferase V belonging to family GT11 (Murray et al., 1997).

Exploration of the FUT1 catalytic pocket highlighted severalresidues of the N-domain that, in addition to the invariant arginine

residue (R366), are potentially important for catalysis (Figure 6A).Mutagenesis studies revealed the critical importance of N184(Figure 6B), a residue strictly conserved in other FUT members offamilyGT37 (Figure5C), andwhich is similarly positionedasN43 inPOFUT1 (Supplemental Figure 6B). Point mutation of D300 toalanine also resulted in complete loss of enzyme activity (Figure6B). In the GDP-fucose model, this acidic position (observed onlyin FUT1 and FUT3 and replaced by an asparagine in other FUTproteins) is at 3.4 Å from the O2 atom of Gal3 and therefore mayplay a role as a general base. Point mutations of S180 and H271residues severely impairedenzymeactivity (theS180AandH271Amutants retained 6.8 and 2.3% activity, respectively; Figure 6B).Thehistidine residue is conserved inall FUTproteinsexceptFUT3,whereas the serine residue is only present in FUT1 and FUT2. Inaddition, H271 closely interacts with D300 side chain and thusmost likely participates in charge relay. The hydrogen bond net-work between D300, H271, S180, and N184 together with R366contributes to create the catalytic pocket of FUT1 (Figure 6A).Particularly, S180 appears to be important for the correct posi-tioning of N184 to establish hydrogen bonds with O2 and O3 ofGal3. Curiously, S180 perfectly superimposes with the proposedcatalytic glutamate residue (E54) in POFUT2. Finally, it is clear thatFUT1 shares common catalytic features with both POFUT1 andPOFUT2 as illustrated in Supplemental Figure 6. The presence ofa catalytic base (D300) correctly positioned to abstract the protonof the nucleophile hydroxyl group of the acceptor in the FUT1active site is in favor of the general SN2 reaction of inverting GTsobserved in POFUT2 (Figure 6C). However, future (mutagenesis)experiments will be necessary to gain further insights into sub-strate/acceptor binding behavior and reaction mechanism ofFUT1.The structures reported here show how FUT1 recognizes its

donor and acceptor substrates and provide information on thereaction mechanism and selectivity of the enzyme. They alsosuggest that FUT1 is organized as a functional dimer in the Golgiapparatus, which may explain its strong preference for polymericacceptor substrates. Further studies exploring the dynamicproperties of FUT1 with longer acceptor molecules are needed toprogress in our understanding of the XyG fucosylation process.

METHODS

DNA Manipulations and Protein Production

TheDNA fragment encoding thestemandcatalytic regions (correspondingto residues 69 to 558 of FUT1 protein sequence) was cloned into the pVT-Bac-His1 transfer vector, generating pVT-Bac-His1-FUT1D68, as pre-viously described (Cicéron et al., 2016). This plasmid harbors the fut1 geneand is preceded by (His)6 and X-press tags used for purification purposes,originating the FUT1D68 protein, named simply FUT1 for clarity. Themutants were generated according to QuikChange II site-directed muta-genesis method (Agilent Technologies), using pVT-Bac-His1-FUT1D68 astemplate, and sense and antisense primers listed in Supplemental Table 3.Mutants were systematically checked by double-strand sequencing.

Each construct was cotransfected with Baculogold AcNPV DNA intoSf9 insect cells (Baculovirus Expression Vector System; Pharmingen) andincubated for 72 h at 27°C. A high-titer virus stock solution (>108 pfu/mL)was used for the infection of High Five cells (Invitrogen). The cells weregrown in Erlen flasks in the serum-free EXCELL405 medium (Sigma-Aldrich) at 27°C, in a shaking incubator at 120 rev/min, for 4 d. Cells and

2360 The Plant Cell

impurities were removed by centrifugation at 13,000g for 30 min and themedium supernatant containing secreted FUT1 was injected througha histidine tag purification column (cOmplete His-Tag; Roche), followed bywashing in 25 mM HEPES buffer, pH 7.4, 500 mM NaCl, and 50 mM im-idazole. Protein was eluted using an imidazole step gradient (up to500 mM). Fractions containing FUT1 protein were pooled and concen-trated, furtherpurifiedbysize-exclusionchromatographyusingaSuperdex200 10/300 GL (GE Healthcare) column and buffer exchanged to 25 mMHEPES buffer, pH 7.4, and 150 mM NaCl. The pure protein was con-centrated up to 7 mg/mL and stored at 220°C until further use. FUT1behaves as a dimer in solution and harbors two N-glycans as verified bysize-exclusionchromatography,dynamic light scattering, andMALDI-TOFanalyses.

FUT1 Enzyme Activity

Fucosyltransferase activity toward tamarind XyG was determined as fol-lows. Enzyme assays were carried in 10 mM HEPES-KOH buffer, pH 7, ina final volume of 100 mL, containing FUT1 (20 nM), 30 mMunlabeled GDP-fucose, 1.5 mM (20 nCi, 45,000 cpm) GDP-[14C]-fucose, and xyloglucanacceptor (from tamarind seeds;Megazyme) at 2.5mg/mL. Reactions wereincubated at 30°C for 30min and 120min and terminated by addition in thereactionmix of 1mL anion exchange AG1x8 resin (1 g resin in 4mL doubledistilled water; 200 to 400 mesh; Bio-Rad), which aims at trapping allnucleotides (GDP and unreacted GDP-fucose). Reaction mix was thencentrifuged 2 min at 13,000g and 600 mL supernatant collected. This laststep was repeated once and the two supernatants pooled and placed intoa counting flask of 5 mL to measure radioactivity incorporation into thexyloglucan polymer. Scintillation liquid (3.8mL) was added to the flask andflasks were counted for 1 min using a Tri-carb 1600TR Packard Bell in-strument (Perkin-Elmer). Control reactions were performed in the absenceof acceptor.

Preparation and Characterization of XLLG

The XLLG acceptor substrate used for soaking experiments was preparedas previously described (Cicéron et al., 2016). Briefly, 100 mg pure XyGfrom tamarind seeds in 1 mL 10 mM sodium acetate were treated with5 units of EG II from Trichoderma reseii (Megazyme) for 18 h at 35°C. Afterheat inactivation of EG II (30min at 70°C), the reactionmix was centrifugedfor 5 min at 13,000g and applied to a syringe filter (0.2 mm from Nalgene).XyG-derived oligosaccharides were then loaded on a HW-40 gel perme-ationcolumn (1m315mm), conditionedwith0.1Mammoniumcarbonate.Refractive index of eluted solvent was monitored and 1-mL fractions werecollected. Three main peaks were identified on the chromatogram andfractions fromthesepeakswerecharacterizedusingMALDI-TOF (Autoflex;Bruker) in reflectron mode according to Lerouxel et al. (2002). Two mi-croliters of fractions containing XyG-derived oligosaccharides weremixedin a 1:1 ratio using a dihydroxybenzoic acid matrix at 2 mg/mL (doubledistilled water/acetonitrile/trifluoracetic acid, 70/30/0.1%). After identifica-tion, fraction #23 containing pure XLLG (peak atm/z = 1409) (SupplementalFigure 7) was collected and lyophilized for experiments.

Crystallization, Diffraction Data Collection, and Processing

Native FUT1 crystallization and crystal characterization are describedelsewhere (Rocha et al., 2016). Suitable x-ray diffracting crystals appearedwithin 3 to 5 days in 100 mM HEPES buffer solutions, pH 7.5 to 7.8, witheither 300 to 500 mM NaCl and 18 to 20% PEG 8000 or 300 to 500 mMNaCH3COO and 20 to 22% PEG 4000. Crystals were cryoprotected inmother liquor solution supplemented with 15% (v/v) ethylene glycol, prior toflash-coolingandstorage in liquidnitrogen.TheFUT1-ligandcomplexcrystalswere obtained after native crystals were soaked in crystallization solutionscontaining eitherGDP-fucoseorGDP+xyloglucanoligosaccharide acceptor

(XLLG). The best FUT1:GDP-fucose protein-ligand data set was obtained byshort incubation (;3 min) of the FUT1 native crystal in a cryoprotective so-lutionof100mMHEPES,pH7.5,250mMNaCl,18%PEG8000,and15%(v/v)ethylene glycol supplemented with 5 mM GDP-fucose. For the soaking ex-periments with GDP and XLLG ligands, the best data set was obtained after;25min incubation of the native crystal in the above solution supplementedwith 10 mM GDP and 7 mg$mL21 XLLG oligosaccharide. Crystals were di-rectlyplungedandstored in liquidnitrogen.Alldiffractiondataweremeasuredat ID29 (de Sanctis et al., 2012) or ID23-2 (Flot et al., 2010) beamlines at theEuropean Synchrotron Radiation Facility (ESRF, Grenoble, France). Diffrac-tion images were processed with XDS (Kabsch, 2010), scaled and mergedwith AIMLESS (Evans and Murshudov, 2013), and the correspondingstructure factors were calculated using cTRUNCATE (Padilla and Yeates,2003) from the CCP4 suite (Winn et al., 2011).

Structure Determination and Refinement

The crystal structure of native enzyme (apo-FUT1) was determined asdescribed previously (Rocha et al., 2016). Briefly, x-ray diffraction data upto 2.9 Å resolution was collected on a single tantalum bromide (Ta6Br12)derivatized crystal, and initial phases were determined at 4.0 Å using the2W-MAD implemented method in SHARP (Bricogne et al., 2003). Phaseswere further improvedwith the programRESOLVE (Terwilliger, 2000) usingthe automatically implemented 4-fold noncrystallographic symmetry av-eraging, solvent flattening, and phase extension procedures up to 2.9 Åresolution.TheRESOLVEoutputmodelwas further inspectedandedited inCOOT (Emsley and Cowtan, 2004) and used as search template for mo-lecular replacement, using a 2.1 Å resolution native data set with theprogram PHASER (McCoy et al., 2007). Iterative model building and re-finement were carried with COOT and PHENIX (Adams et al., 2010) untilmodel completion.

The FUT1:GDP:XLLG structure was obtained after rigid body re-finement with REFMAC5 (Murshudov et al., 2011) of the apo-FUT1monomers against the 2.2 Å resolution FUT1:GDP:XLLG data set. Readilyinterpretable electron density maps were produced and clear well-definedresidual density was visible for a GDP molecule nestled in all four mole-cules. Extra electron density that could accommodate the XLLG acceptorwas also found in the residual maps for two protein monomers.

The phases for the FUT1:GDP-fucose structure were also retrieved byrigid body refinement of apo-FUT1 with the 2.5 Å resolution protein-liganddata set. Inspection of the initial electron density maps revealed electrondensity that could accommodate only theGDPmolecule. Since no densitywas found for the fucose, and theGDP ligandoccupied thesameposition inthe higher resolution FUT1:GDP:XLLG model, no further analysis of theFUT1:GDP-fucose structure was performed. Further steps of protein-ligand model refinement were performed with COOT and PHENIX. Themolecular stereochemistry of FUT1 structures was evaluated using theprogram MOLPROBITY (Chen et al., 2010). Intermolecular contacts andcrystallographic packing were retrieved with PISA (Krissinel and Henrick,2007), and structural comparisons among themodelswereperformedwithSSM (Krissinel and Henrick, 2004). Images were prepared with PYMOL(The PyMOLMolecular Graphics System, version 1.7.4, Schrödinger LLC).

Crystal Characterization and Validation of FUT1 Models

Upon successive crystal optimizations and data collection sessions, datasets of apo-FUT1 and FUT1:GDP:XLLG ternary complex reached finalresolutions of 2.1 and 2.2 Å, respectively. Detailed crystal and diffractiondata statistics are given in Supplemental Table 1. Crystals belonged tospace group P21 with similar cell dimensions and contained four in-dependent molecules in the asymmetric unit, which were organized intotwo distinct dimeric structures designated as A-B and C-D (SupplementalFigure 2). Overall, the entire length of the peptide chains showed goodelectron density, except for the small missing segments located at the

Xyloglucan Fucosyltransferase Structure 2361

N- and the C-domains (Na3-Nb1, Na5-Nb3, Ca2-Cb2, and Cb3-Ca4loops) or some residue side-chains exposed to the solvent that did notshowwell-defined density for all the atoms. Globally, the apo-FUT1modelcontained 1809 out of the 2084 residues (4 chains 3 31 residue tag plus490 residues). In all four monomers, the N-terminal 31-residue tag was notvisible and thus was not included in the model. Also, some loop regions(namely, S69-R79, D257-G260, V399-N406, and G450-K457 for chain A;S69-S94, E163-G167, D257-E259, H404-N406, and G450-K457 for chainB; S69-N93, T258-E259, E400-N406, and G450-K457 for chain C; S69-A83, D166-D168, I256-E259, H404-N406, and G450-K457 for chain D)were not visible in the electron density maps and therefore were omitted inthe finalmodel.When appropriate, ions and solventmoleculeswere addedto themodel. The apo-FUT1was refined toRwork/Rfree of 17.73/21.51%andthe final model contained 96.7, 3.1, and 0.2% of the residues within thepreferred, allowed, or outlier regions of the Ramachandran diagram(Ramachandran et al., 1963), respectively (see Supplemental Table 2 forrefinement and model quality statistics).

The FUT1:GDP:XLLG model contained 1801 out of the 2084 totalresidues. Similarly to the apo-FUT1 structure, some residues were notvisible in electrondensitymapsandwereomitted from themodel: the initial31-residue tag for each chain, and S69-N91, D166-G167, D257-G260,V399-H404, and Q452-K456 for chain A; S69-S94, E163-G167, D257-E259, H404-N406, and T454-K456 for chain B; S69-N93, D257-E259,E400-N406, and Y451-K456 for chain C; S69-N93, D166-D168, I256-G260, H404-N406, and E455-K456 for chain D. Moreover, four GDPligands (one per monomer) and two XLLG molecules (one per dimer) weremodeled intowell-defined electron density. Several solventmolecules andions were also added to the model (see Supplemental Table 2 for furtherdetails). The FUT1:GDP:XLLG final model refined to Rwork/Rfree values of17.90/21.49% contained 96.6, 3.2, and 0.2% of residues within the pre-ferred, allowed,andoutlier regionsof theRamachandranplot, respectively.

Modeling of GDP-Fucose

The structure of the GDP-fucose was built by superposition of the GDPstructure observed in FUT1 and the structure of the GDP-fucose observedin POFUT1 (Lira-Navarrete et al., 2011). Only atoms of the diphosphatebridges were aligned to add the fucose ring to GDP, so that the GDPconformation and position were maintained in the complex. Only minimalmodifications were made afterwards tomaintain the features of the crystalstructure.Hydrogenswereaddedusing theSchrödingerSuite (http://www.schrodinger.com). Steric clashes between the fucose ring and few sur-rounding residues of both the protein and the XyGacceptorwere solved byenergy minimization using the Sybyl-X suite (http://tripos.com). Standardparameters were used for energy calculation, including parameters fromthe TRIPOS force field. The final model was analyzed through comparisonof theactive sitesof the fourmonomers (A,B,C, andD) in theAU.Theactivesites of monomers B and C (with a bound acceptor molecule) were con-sidered for mechanistic consideration of FUT1 enzyme.

Accession Numbers

Sequence data from this article can be found the UniProt KB databaseunder the following accession numbers: Q9SWH5, O81053, Q9CAZ1,Q9SJP2, Q9SJP4, Q9XI80, Q9XI81, Q9XI78, Q9XI77, and Q9SJP6 forFUT1 to FUT10, respectively. The structures of apo-FUT1 and FUT1:GDP:XLLGhavebeendeposited in theProteinDataBank (PDB) under accessionnumbers 5KOP and 5KOR, respectively. The PDB codes of POFUT1 andPOFUT2 structures used in this work are 3ZY5 and 4AP6, respectively.

Supplemental Data

Supplemental Figure 1. Overall Architecture of the Classical GT-BFold and Variants in Crystal Structures of Fucosyltransferases.

Supplemental Figure 2. Asymmetric Unit Composition of FUT1Crystals.

Supplemental Figure 3. Structure-Based Sequence Alignment of theMost Conserved Regions in FUT1 and Its Closest Structural Homo-logs.

Supplemental Figure 4. View of the Active Sites in Monomers A and Bof Apo-Fut1.

Supplemental Figure 5. Superimposition of the FUT1:GDP:XLLGCrystallographic Structure with the Modeled GDP-Fucose.

Supplemental Figure 6. Comparison of the Active Sites of FUT1 withPOFUT1 and POFUT2.

Supplemental Figure 7. MALDI-TOF Analysis of XLLG Preparation

Supplemental Table 1. Data Collection and Crystallographic Statis-tics for FUT1 Crystals.

Supplemental Table 2. Refinement and Model Quality Statistics forFUT1 Structures.

Supplemental Table 3. Oligonucleotides Used for PCR Mutagenesis.

ACKNOWLEDGMENTS

We thank Ken Keegstra for his critical reading of the manuscript andinsightful comments. This work was supported by the CNRS and theUniversity Grenoble Alpes. F.C. received a PhD fellowship from UGA.Access to the ESRF Structural Biology beamlines is gratefully acknowl-edged.

AUTHOR CONTRIBUTIONS

C.B. and O.L. conceived the project and designed research. J.R., F.C.,D.d.S., M.L., V.C., and O.L. performed research. C.B., J.R., and O.L.discussed results. C.B. and J.R. wrote the manuscript.

Received July 7, 2016; revised August 29, 2016; accepted September 12,2016; published September 16, 2016.

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2364 The Plant Cell

DOI 10.1105/tpc.16.00519; originally published online September 16, 2016; 2016;28;2352-2364Plant Cell

and Christelle BretonJoana Rocha, Félix Cicéron, Daniele de Sanctis, Mickael Lelimousin, Valérie Chazalet, Olivier Lerouxel

Insight into Xyloglucan Fucosylation FUT1 Reveals a Variant of the GT-B Class Fold and ProvidesArabidopsis thalianaStructure of

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