Structure of Xyloglucan Xylosyltransferase 1 Reveals Simple Steric Rules That Define Biological Patterns of Xyloglucan Polymers

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Structure of xyloglucan xylosyltransferase 1 reveals simple steric rules that define biological patterns of xyloglucan polymers

Alan T. Culbertsona, Jacqueline J. Ehrlicha, Jun-Yong Choeb, Richard B. Honzatkoa, and Olga A. Zabotinaa,1

aRoy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011; and bDepartment of Biochemistry and Molecular Biology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064

Edited by Kenneth Keegstra, Michigan State University, East Lansing, MI, and approved April 27, 2018 (received for review January 19, 2018)

The plant cell wall is primarily a polysaccharide mesh of the most substrates, including, carbohydrates, proteins, and lipids (12). abundant biopolymers on earth. Although one of the richest Amino acid sequences in the Carbohydrate Active Enzyme Da- sources of biorenewable materials, the biosynthesis of the plant tabase fall into 105 families of GTs (13, 14). Available structures polysaccharides is poorly understood. Structures of many essential indicate most GT families adopt one of two folds, GT-A or GT- plant glycosyltransferases are unknown and suitable substrates B, although a rarer GT-C fold has been proposed (12). The GT- are often unavailable for in vitro analysis. The dearth of such A fold has two Rossmann-like domains that form a central information impedes the development of plants better suited for β-sheet, each face of which is covered by α-helices. These are industrial applications. Presented here are structures of Arabidop- typically metal-dependent enzymes that require an Asp-X-Asp sis xyloglucan xylosyltransferase 1 (XXT1) without ligands and in motif for metal coordination (15, 16). GT-B folds also have two complexes with UDP and cellohexaose. XXT1 initiates side-chain Rossmann-like domains, less tightly associated than those of GT- extensions from a linear glucan polymer by transferring the xylo- A folds, with the active site located between domains. GTs are syl group from UDP-xylose during xyloglucan biosynthesis. XXT1, also classified by the stereochemistry of the glycosidic bond in a homodimer and member of the GT-A fold family of glycosyl- the product (inverted or retained) relative to that of the sub- transferases, binds UDP analogously to other GT-A fold enzymes. strate (12). The catalytic mechanism of inverting GTs likely follows the single displacement mechanism of inverting glycosyl Structures here and the properties of mutant XXT1s are consistent hydrolases (12, 17). The catalytic mechanism of retaining GTs, with a SN -like catalytic mechanism. Distinct from other systems is i first proposed as a double displacement mechanism similar to the recognition of cellohexaose by way of an extended cleft. The retaining glycosyl hydrolases, has fallen into disfavor due to the XXT1 dimer alone cannot produce xylosylation patterns observed absence of a suitably placed catalytic base and the failure to trap for native xyloglucans because of steric constraints imposed by the a glycosyl-enzyme intermediate. Instead, retaining GTs may acceptor binding cleft. Homology modeling of XXT2 and XXT5, the employ a SNi-like mechanism that consists of the acceptor sub- other two xylosyltransferases involved in xyloglucan biosynthesis, strate approaching from the same face as the leaving group with reveals a structurally altered cleft in XXT5 that could accommodate an oxocarbenium-ion intermediate (18–20). Although structural a partially xylosylated glucan chain produced by XXT1 and/or information is abundant for glycosyltransferases (21), structural XXT2. An assembly of the three XXTs can produce the xylosylation information specifically for GTs involved in plant cell wall patterns of native xyloglucans, suggesting the involvement of an polysaccharide biosynthesis is available only for xyloglucan organized multienzyme complex in the xyloglucan biosynthesis. Significance glycosyltransferases | plant cell wall | xyloglucan

The recalcitrant nature of the plant cell wall presents a signif- lant cell walls consist of cellulose, hemicellulose, pectin, and icant challenge in the industrial processing of biomass. Poor Plignin, all of which confer mechanical properties to plant understanding of plant polysaccharide biosynthesis impedes structures, and are important for shape and development. Plant efforts to engineer cell walls susceptible to efficient and un- cell walls represent the largest pool of renewable carbohydrate natural pathways of degradation. Despite numerous genetic and the potential to support numerous industrial applications in and in vitro studies of the xyloglucan xylosyltransferases bioenergy and biomaterials (1, 2). The complex structure of plant (XXT1, XXT2, and XXT5), the specific roles of each in the xylo- lignocellulosic biomass resists enzymatic and microorganism sylation of the xyloglucan backbone is unclear. On the basis of degradation (3). Engineering a biologically viable plant suscep- steric constraints imposed by the active-site cleft of structures tible to enzymatic or nonbiological degradation requires a presented here, we propose a multienzyme complex capable of complete understanding of plant cell wall polysaccharide bio- producing the xylosylation patterns of native xyloglucans. This synthesis and structure. model significantly extends our limited understanding of Xyloglucan (XyG) is the most abundant hemicellulose in the branched polysaccharide biosynthesis. primary cell wall of dicotyledonous plants and has many proposed structural and regulatory functions (4–7). XyG consists of Author contributions: A.T.C., R.B.H., and O.A.Z. designed research; A.T.C. and J.J.E. per- a 1,4-β-linked glucan backbone branched with various glycosyl formed research; A.T.C., J.-Y.C., and R.B.H. performed experimental phasing; A.T.C., residues depending on species or tissue (8). The nomenclature R.B.H., and O.A.Z. analyzed data; and A.T.C., J.-Y.C., R.B.H., and O.A.Z. wrote the paper. for XyG structure is as follows: G represents an unbranched The authors declare no conflict of interest. glucose unit, whereas X, L, and F are glucosyl units with Xyl, This article is a PNAS Direct Submission. Gal-Xyl, or Fuc-Gal-Xyl side chains, respectively (9). Arabidopsis Published under the PNAS license. XyG consists of a glucan backbone branched with 1,6-α-linked D- Data deposition: The atomic coordinates and structure factors have been deposited in the Xyl residues, resulting in XXXG-type pattern, which can be Protein Data Bank, www.wwpdb.org (PDB ID codes 6BSU, 6BSV, and 6BSW). further decorated (10, 11). 1To whom correspondence should be addressed. Email: [email protected]. Glycosyltransferases (GTs) catalyze the formation of glyco- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sidic bonds by transferring a sugar moiety from an activated 1073/pnas.1801105115/-/DCSupplemental. donor, typically a nucleotide sugar, to a variety of acceptor Published online May 21, 2018.

6064–6069 | PNAS | June 5, 2018 | vol. 115 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1801105115 Downloaded by guest on September 28, 2021 ABXXT2 when expressed in Drosophila cells (32). The structure of the enzyme (in crystals of space group P212121) was solved by single-wavelength anomalous diffraction (SAD), using data from 2+ a crystal derivatized with K2HgI4. The complex of UDP/Mn (hereafter, the binary complex) and the complex of cellohexaose/ + UDP/Mn2 (hereafter the ternary complex) were formed by li- + gand soaks. XXT1 exhibits highest activity with Mn2 (32), which was included in all ligand soaks. A second crystal form of the apoenzyme (space group C2221), diffracting to 1.5-Å resolution, was solved by molecular replacement. Given its superior resolution, the apoenzyme in space group C2221 is reported in SI Appendix, Table S1. Regardless of crystal form, the asymmetric CDunit has two subunits of XXT1, and for each, residues 45– 115 from the N terminus and residues 454–460 from the C terminus are without electron density. The purified protein has an observed mass consistent with that expected for residues 45–460. Finally, a substantial void exists in both crystal forms that could accommodate 70 additional residues. XXT1 adopts a GT-A fold with a central β-sheet having both faces covered by α-helices (Fig. 1 A and C). The central β-sheet contains strands β2, β1, β3, β6, β5, and β7, all of which are par- Fig. 1. Structure overview of XXT1. (A) XXT1 monomer (chain A) colored allel except for strand β6 (Fig. 1 A and C). The loops extending blue to red from N to C terminus, respectively. (B) Secondary and tertiary from this central β-sheet and surrounding α-helices define the structure of XXT1. Names of α-helices and β-strands correspond to those in A. active site of XXT1, containing the Asp-X-Asp motif (site of + (C) XXT1 dimer with UDP and cellohexaose. Monomers are shown in green Mn2 binding), donor substrate binding site, and acceptor and yellow. UDP binds to both monomers, whereas cellohexaose binds to binding site (Fig. 1A). The active site is a cleft roughly 13 Å wide only one monomer. (D) View down the symmetry axis of the dimer, 90° and 30 Å long (Fig. 1A). rotation of the molecule shown in C. XXT1 Forms a Dimer. XXT1 forms a dimer with an interface of 2 fucosyltransferase 1 (FUT1), which adds fucose to the terminal roughly 2,900 Å (PDBe PISA; www.ebi.ac.uk/pdbe/pisa/). position of XyG side chains (22, 23). XXT1 dimer has a mass of 96.7 kDa based on amino acid se- Xylosylation of the 6-hydroxyl group of glucose, catalyzed by quence relative to 102.7 kDa determined by gel filtration against xyloglucan xylosyltransferases (XXTs), is the first step in building protein standards of known mass. The high value from gel fil- branches on the XyG backbone (8, 24). XXTs are type II tration is consistent with an increased radius of gyration due to the aforementioned loose stem region of 70 residues (SI Ap- transmembrane enzymes, having a short cytosolic N-terminal pendix A B region, a transmembrane domain, a stem region, and a large , Fig. S1 and ). The interface between subunits in- volves the side chains of Leu171, Leu172, Ile175, Ile179, C-terminal catalytic domain localized in the Golgi lumen (25– PLANT BIOLOGY and Tyr191 from helix α2 and strand β2 from each subunit (SI 27). Three XXTs xylosylate the XyG backbone both in vivo (28– Appendix, Fig. S1E). Surrounding this hydrophobic core are 30) and in vitro (25, 31–34). These enzymes putatively adopt GT-A folds, have products that retain the stereochemistry, and belong to the GT34 family. Family GT34 contains seven proteins Arabidopsis from (31), including xyloglucan xylosyltransferases, ACTrp225 Ala158 galactomannan 1,6 galactosyltransferases and 1,2 galactosyl-

transferases (CAZy: www.cazy.org). Enzymatic action of XXTs Gly202 in vitro employ short glucans, such as cellohexaose or cello- Gly156

pentaose, due to the low solubility of long glucan chains. XXT1 3.173.17 Lys382 (and XXT2) primarily adds the first xylosyl residue to the fourth Ser228 glucose residue from the reducing end of cellohexaose. XXT1 3.213.21

(and XXT2) then adds a xylosyl residue to the third or fifth Val379 2.872.87 3.02 glucose from the reducing end of cellohexaose (32). Phe203 2.912.91 Presented here are crystal structures of XXT1 without ligands, 2.70 2.95 2.102.10 a binary complex with UDP, and ternary complex with UDP and 2.69 2.102.10 Gly380 3.163.16 Mn 2.21 cellohexaose. This is the first structure from the GT34 family and 2.122.12 3.173.17 2.092.09 2.50 only the second crystal structure of a plant cell wall glycosyl- B 3.08 transferase (the first being of FUT1). XXT1 is a homodimer in solution and crystal. Structural comparisons with retaining GT-A His377 fold glycosyltransferases and directed mutations support a SNi- Asp229 like catalytic mechanism. Moreover, the crystal structure and homology models indicate similar steric requirements for glucan interactions with XXT1 and XXT2, but different requirements for XXT5, suggesting the participation of XXT1 (or XXT2) with XXT5 in achieving the in vivo pattern of xylosyl transfer to the linear glucan. Results Fig. 2. Bound UDP in two conformations. (A) Bent conformer, cellohexaose Overall Structure of XXT1. Protein expression of the XXT1 stem – absent. (B) Extended conformer, cellohexaose present. Colors in A and B region and catalytic domain (residues 45 460) was performed in are as follows: Tan, XXT1 secondary structure; blue, nitrogen atoms; red, human embryonic kidney cells (35). SDS/PAGE analysis reveals oxygen atoms; cyan, carbon atoms of ligands; orange, phosphate atoms; + a double band that is routinely observed in our preparations of purple, Mn2 .(C) Interaction map for the extended conformer of UDP from XXT1 (SI Appendix, Fig. S1D) and also observed for XXT1 and LigPlot+ (45).

Culbertson et al. PNAS | June 5, 2018 | vol. 115 | no. 23 | 6065 Downloaded by guest on September 28, 2021 AB the sugar residue folds over the phosphoryl groups. An analogous conformation for UDP-xylose is present in the structure of XXYLT1 (PDB ID 4WNH and SI Appendix, Fig. S4B) and LgtC (18, 19). That model for UDP-xylose, when superimposed on the extended UDP molecule in XXT1, fits the active site, with the xylosyl residue occupying a pocket of uniform/unstructured electron density. Energy minimization led to the model in Fig. 3, which retains the conformations of UDP, cellohexaose, and active-site side chains (SI Appendix, Fig. S4A). The 3-hydroxyl group of xylose hydrogen bonds with Gln319 and Thr269, and the 4-hydroxyl group hydrogen bonds with Lys207 and Asp318 (Fig. 3). Phe203, which has a high B parameter in crystal structures, makes a favorable contact with atom C5 of the xylosyl residue, a contact that would discriminate against a hexose sugar. Fig. 3. Model of bound UDP-xylose. (A) Position of UDP-xylose with respect Acceptor Substrate Binding. The ternary complex of XXT1 has to cellohexaose. The arrow marks the 6-hydroxyl group of the fourth glucose only one monomer of the dimer with bound cellohexaose (Fig. 1 from the reducing end. (B) Distances (in angstroms) between selected atoms C D of XXT1 and xylosyl group of UDP-xylose. Colors are defined in Fig. 2. and ). Access to the active site in the nonoccupied monomer is limited due to packing contacts. The cellohexaose molecule is covered by strong electron density and has B parameters signif- electrostatic interactions and hydrogen bonds obeying the two- icantly lower than those of UDP (SI Appendix, Fig. S3A). The fold symmetry of the dimer: Gln432-NE2 to the backbone car- interactions of XXT1 with the glucosyl residue of cellohexaose bonyl of Leu119; Asp168-OD1 to His169-ND1; the backbone defines six subsites (hereafter subsites 16). Subsite 1 binds the amide of Tyr191 to Lys176-NZ; Tyr414-OH to His217-NE2; reducing end of cellohexaose. The 6-hydroxyl of the fourth glu- Glu188-O1 to Lys451-NZ; and Glu219-OE1 to His447-NE2. cosyl residue projects into the active site of XXT1 (Fig. 4), in Interacting residues at the subunit interface of the dimer are agreement with the predominant modification of cellohexaose at identical in XXT1 and XXT2, suggesting the possibility of het- its fourth glucosyl residue in solution (32). Glucose residues 3– erodimer formation (SI Appendix, Fig. S2). Heterodimer for- 5 of cellohexaose make direct hydrogen bonds at subsites 3–5, mation would likely have no major impact on xylosylation respectively. Conversely, the first, second, and sixth glucosyl pattern due to the predicted functional redundancy of XXT1 and residues have water-mediated or hydrophobic interactions at XXT2 (28, 32). Although twofold molecular symmetry is obeyed subsites 1, 2, and 6, respectively (Fig. 4). Glucosyl residues at the across the subunit interface, cellohexaose binds to only one of ends of cellohexaose have weak electron density, whereas resi- two active sites of the UDP/cellohexaose complex. The basis for dues 2–4 have strong electron density with clearly defined 6- this asymmetry in the binding of cellohexaose is provided below. hydroxyl groups that unambiguously determine the orientation of cellohexaose and the placement of its reducing and non- Donor Substrate Binding. Binary (UDP) and ternary (UDP and reducing ends. The strong electron density of 6-hydroxyl groups cellohexaose) structures of XXT1 were obtained by soaking of cellohexaose suggests a single allowed orientation of the XXT1 crystals with the substrates, along with MnCl2, for 2 d; the bound glucan to XXT1 (Fig. 4B and SI Appendix, Fig. S3A). structures were refined against data to a resolution of 2.4 Å and Interestingly, bound glucan chains in the active sites of the 2.1 Å, respectively (SI Appendix, Table S1). In both the binary 2+ XXT1 dimer point in opposite directions. Thus, both subunits of and ternary complexes, Mn is coordinated through a mono- the XXT1 dimer would unlikely xylosylate the same glucan dentate interaction with Asp227, bidentate interaction with chain, but more likely act on two separate chains. The glucosyl Asp229, and NE2 of His377. The UDP molecules have different residue at subsite 3 hydrogen bonds with His346 and stacks with conformations in the presence and absence of cellohexaose. The + extended conformer in the ternary structure coordinates Mn2 through one oxygen atom from each of the α- and β-phosphoryl groups (Fig. 2B). The bent conformer in the binary structure + AC coordinates Mn2 through one oxygen atom of the β-phosphoryl Trp254 Tyr344 A 2.92 group and a nitrate anion (Fig. 2 ). Interactions are identical for 2.73 Tyr348 the bent conformer of UDP in the active site of the ternary complex that lacks cellohexaose and the active sites of the binary 2.682.68 2.762.76 2.92 His346 β 2.73 complex. The -phosphoryl group of the extended UDP con- 2.73 2.892.89 2.67 Met257 3.013.01 former hydrogen bonds with the 6-hydroxyl group and the 2- 2.882.88

hydroxyl group of the fourth and fifth glucose units, respec- 2.94 2.312.31 Ile265 3.35 3.023.02 2.702.70 tively, from the reducing end of bound cellohexaose. In addition, 2.80 2.892.89 2.722.72 Asn268 atom NZ of Lys382 hydrogen bonds with oxygen atoms of the 3.093.09

α β 2.882.88 - and -phosphoryl groups of the extended conformer (Fig. 2). 3.303.30 Leu267 2.932.93 2.902.90 The extended conformation of UDP likely approximates the B Tyr390 catalytically productive binding of UDP-xylose and is similar to Gln319 2.712.71 nucleotide conformations in other retaining GT-A fold glyco- 2.632.63 2.692.69 Asp389 2.70 syltransferases (18, 19). Additionally, the bent conformation may Pro312 3.19 UDP

indicate that interactions with XXT1 and UDP are weak, and Trp264 that most of the dominant interactions are between XXT1 and Gly380 Lys382 the xylose of UDP-xylose. Val379

Modeling of UDP-Xylose. Attempts to cocrystallize UDP-xylose with XXT1 or soak UDP-xylose into preformed crystals did not Fig. 4. Acceptor substrate binding site in the ternary complex. (A) Residues reveal a bound donor substrate. Instead, UDP-xylose was mod- of XXT1 in proximity to cellohexaose. (B) Cellohexoase with electron density eled into the active site of the ternary structure. GT structures from an omit map contoured at 2σ. Colors are defined in Fig. 2. (C)In- typically have similar bound UDP-sugar conformations in which teraction map for cellohexaose from LigPlot+ (45).

6066 | www.pnas.org/cgi/doi/10.1073/pnas.1801105115 Culbertson et al. Downloaded by guest on September 28, 2021 Tyr348 (Fig. 4). Glucosyl residue 5 hydrogen bonds with Asp389, UDP (less than 20% of wild-type) are Lys382Ala, Asp317Ala, Lys382, and an oxygen atom of the β-phosphoryl group of UDP. Asp318Ala, and Gln319Ala. Lys382 hydrogen bonds with oxygen Xylosyl extensions from the 6-hydroxyl groups of glucosyl atoms of the α- and β-phosphoryl groups of UDP (extended residues at subsites 1, 3, 5, or 6 should not introduce steric conformer), whereas the remaining residues interact with the conflicts (SI Appendix, Fig. S5). The 6-hydroxyl groups at subsites xylosyl group of modeled UDP-xylose. Evidently, mutations 3 and 5 are pointed away from the acceptor cleft and are solvent proximal to the xylosyl group of the donor substrate have the exposed (SI Appendix, Fig. S5A). The 6-hydroxyl groups at sub- greatest impact on the rate of product formation. To confirm sites 1 and 6 have space to accommodate a xylosyl adduct (SI activity, the Asp317Ala, Asp318Ala, and Gln319Ala mutants Appendix, Fig. S5 B and C). Moreover, relatively low levels of were assayed with sixfold higher protein concentration, demon- electron density associated with glucose units at subsites 1 and strating clear UDP production (SI Appendix, Fig. S6B) and 6 suggests weaker binding at these subsites for an extended xylosylated cellohexaose formation was confirmed by MALDI- glucan chain (Fig. 4 and SI Appendix, Fig. S3A). In contrast, a TOF analysis (SI Appendix, Fig. S6 F–H). xylosyl extension at subsite 2 would overlap with the side chains of His252, Ile351, and Tyr344, suggesting that xylose transfer to Catalytic Mechanism of XXT1. Inverting GTs utilize a single- glucose unit N blocks subsequent binding necessary for xylose displacement mechanism similar to that of inverting glycosyl transfer to glucose unit N + 2. hydrolases (12, 17). The catalytic mechanism for retaining GTs, however, is unclear. Retaining glycosyl hydrolases utilize a Mutations of Active Site Residues. XXT1 wild-type and mutant double-displacement mechanism in which a catalytic base first enzymes were assayed using the UDP-GLO assay (Promega) to inverts the anomeric stereochemistry of the sugar, forming a measure UDP formation with 20-min reactions (Fig. 5A). These glycosyl-enzyme intermediate (12, 17). A second nucleophilic assays revealed comparable activities for the wild-type and attack at the anomeric carbon of the glycosyl-enzyme in- Ser228Ala enzymes, but activity levels for other mutant enzymes termediate by the hydroxyl group of the acceptor restores the were statistically indistinguishable from an enzyme-free buffer original anomer. For GTs however, a suitable catalytic base is (blank). Twenty-hour reactions were then performed to better not evident in structures (18, 36, 37) and efforts to trap a gly- determine the loss of activity of the least active mutant enzymes. cosyl-enzyme intermediate have been unsuccessful (38). Hence, The 20-h reaction resulted in roughly fourfold higher UDP some have suggested a SNi-like mechanism for retaining GTs as SI Appendix A production for wild-type XXT1 ( ,Fig.S6 ). an alternative (12, 18). In the SNi-like mechanism an oxo- XXT1 assays lacking UDP-xylose resulted in no xylosylation of carbenium-ion intermediate forms; however, the acceptor ap- cellohexaose (SI Appendix,Fig.S6A and D). Similarly, proaches the same face of the oxocarbenium-ion intermediate as XXT1 assays lacking cellohexaose had no detectable UDP the leaving group. This mechanism has gained support from compared with assays lacking XXT1, indicating XXT1 does not studies in quantum/molecular mechanics (39), kinetic isotope hydrolyze UDP-xylose (SI Appendix, Fig. S6A). Mutant enzymes effects (20), and crystallography (snapshots along the reaction Ser228Ala and Asn268Ala have UDP levels comparable to those pathway) (19). of the wild-type enzyme (Fig. 5B). Mutations of residues that The XXT1 active site has no nucleophile positioned suitably + coordinate Mn2 in the wild-type enzyme (single mutant en- for a double-displacement mechanism. Asp317 and Asp318 are zymes Asp229Ala and His377Ala, and the double mutant en- 5.4 and 5.7 Å away, respectively, from the anomeric carbon in the zyme Asp227Asn/Asp229Asn), produce 25–30% of the wild-type UDP-xylose model. Invariant conformations of Asp317 and levels of UDP. Mutant enzymes producing the lowest levels of Asp318 over all crystal structures and low B parameters are not PLANT BIOLOGY indicative of a region predisposed to conformational change, yet mutations at positions 317 and 318 have among the largest impacts on activity (Fig. 5). In the structure of XXYLT1 (19), a A hydrogen bond between Gln330 and the 2-hydroxyl group of the xylosyl could in principle stabilize the positive charge of an oxocarbenium ion during the SNi-like reaction mechanism (19). Gln319 of XXT1 assumes a position analogous to Gln330 in XXYLT1 (SI Appendix, Fig. S4B), 4.7 Å from the anomeric carbon atom of modeled UDP-xylose, forming a hydrogen bond with the 3-hydroxyl group of the xylosyl residue. The Gln319Ala mutation has one of the largest impacts on activity (Fig. 5).

Homology Modeling of XXT2 and XXT5. Efforts to purify and con- centrate XXT2 and XXT5 to levels sufficient for crystallization B have been unsuccessful; however, the structure of XXT1 leads to homology models of XXT2 and XXT5 that have no major de- partures from the backbone structure of XXT1 (SI Appendix, Fig. S7A). XXT1, XXT2, and XXT5 have nearly identical positions for all residues in the active site with one notable differ- ence: XXT1 puts an isoleucine residue at subsite 2 proximal to the 6-hydroxyl group of the second glucosyl from the reducing end of cellohexaose, whereas XXT5 has a glycine residue in the corresponding location (SI Appendix, Fig. S7 C and E). The glycine in XXT5 likely allows a xylosyl moiety at subsite 2, suggesting that XXT5 (in contrast to XXT1 and XXT2) can transfer a xylosyl moiety to glucose N when the N + 2 glucosyl toward the reducing end has been xylosylated (SI Appendix, Fig. S7 B–E). Fig. 5. Activity of XXT1 mutants and wild-type (WT) enzymes. (A) Twenty- Additionally, the XXT1 surface contains a region rich in acidic minute activity assays. (B) Twenty-hour activity assays. Each datum repre- residues (Glu357, Glu358, and Glu361) and one rich in basic sents the average of three assays measuring the production of UDP from residues (Lys419, Arg425, and Lys427; SI Appendix, Fig. S8 A starting concentrations of 2 mM UDP-xylose, 1 mM cellohexaose, and 2 mM and B). These residues are conserved in XXT1, XXT2, and SI Appendix MnCl2, in 50 mM Tris pH 7.4 and 75 mM NaCl. Error bars are drawn at one SD. XXT5 ( , Fig. S2). Matching the acidic region of one Blank represents activity assay with enzyme-free buffer. XXT1 dimer to the basic region of another XXT2 dimer results

Culbertson et al. PNAS | June 5, 2018 | vol. 115 | no. 23 | 6067 Downloaded by guest on September 28, 2021 in a feasible dimer-to-dimer interface (SI Appendix, Fig. S8C). A the partial redundancy in function of XXT1 and XXT2 as glucan chain would thread from one active site to that of its demonstrated by in vivo (28, 29) and in vitro (32) are consistent neighboring dimer, suggesting the possibility of coordinated with similar substrate specificities, steric limitations, and modes xylosyl transfers to the glucan chain. of action. The residue that would be in steric conflict with a xylosylated glucose at subsite 2 is isoleucine in XXT1 and XXT2, Discussion but is glycine in XXT5 (SI Appendix, Fig. S7). Hence, it is likely Of all plant cell wall GTs, only the structure of FUT1 is known that the N + 2 rule does not apply to XXT5 (Fig. 6). (22, 23). The crystal structure of XXT1 is the first instance of a A model for xylosylation of the glucan chain in vivo must GT34 family member and the second example of a GT involved satisfy the following assumptions and observations. First, cellu- in plant cell wall biosynthesis. XXT1 and FUT1 represent dif- lose synthase-like C4 (CSLC4) probably functions like cellulose ferent folds, GT-A for XXT1 and GT-B for FUT1. In addition, synthase, adding 1,4-β-linked glucosyl residue onto the non- XXT1 employs a large cleft in acceptor binding, whereas reducing end of the growing glucan chain (40, 41). Hence, the FUT1 does not (22, 23). The twofold symmetry of XXT1 active reducing end of the glucan would emerge first from the synthase. site clefts would require the glucan chain to bend 180° for both Second, xxt5 knockout plants exhibit two- to fourfold increases in active sites to modify a single glucan chain. Given the structural GXXG-type xyloglucan and a threefold decrease in XXXG-type properties of the glucan chain, such a bend is unlikely. Instead, xyloglucan (29). Third, the glucan chain binds in a specific ori- XXT1 probably acts on separate glucan chains, nearly parallel entation to XXT1, and on the basis of homology models, the and in opposite orientations as defined by their reducing and same orientation for XXT2 and XXT5 (this study). Fourth, nonreducing ends. XXT1, XXT2, and XXT5 have similar six-subunit clefts (this XXT1 is catalytically active on cellopentaose and cellohexaose study). Fifth, XXT1 and XXT2 adhere to the N + 2 rule, whereas with xylosylation primarily on glucose residue 4 (31, 32). The XXT5 does not (this study). Sixth, XXT1 interacts with XXT2, ternary complex (PDB ID 6BSW) directly supports xylosylation and XXT2 interacts with XXT5, but XXT1 does not interact of cellohexaose at the fourth glucose residue from the reducing strongly with XXT5 (42−43). Finally, the glucan is a helix of end (Fig. 2). The addition of a second xylosyl moiety requires cellobiose repeats, with an angular displacement (rotation) monoxylosylated cellohexaose to disassociate from XXT1, rotate of 51.4° per cellobiose unit (44). The second glucosyl residue of about the axis of the glucan by 180°, and rebind, shifted by one cellobiose has an additional angular offset of 180° relative to the glucosyl residue. Shifting (after rotation) by one glucosyl residue first glucose. Fig. 6 presents a plausible sequence of events occupies subsites 1–5 or subsites 2–6. Binding of 6GGXGGG1 at subsites 1–5 should generate the product GXXGGG , whereas during xyloglucan biosynthesis that accommodates the preceding 6 1 observations/assumptions. Emerging glucosyl residues from the binding at subsites 2–6 should generate the product 6GGXXGG1 (SI Appendix, Fig. S9). Cavalier and Keegstra (32) observed both glucan synthase thread into XXT1 first, followed by XXT2, and then XXT5, the order determined by the observed binding products, with 6GGXXGG1 preferred. Given that subsite 2 can- – – not accommodate a xylosyl adduct, XXT1 cannot transfer a preferences of the XXT protein protein interactions (XXT1 XXT2 and XXT2–XXT5) and the recognition that XXT5 must xylosyl moiety to 6GGXXGG1 to produce 6GXXXGG1,termi- nating further modification; however, XXT1 can transfer a xylosyl come last to produce the XXXG repeat. The glucan chain in this model must advance by four glucosyl residues with each cycle. moiety to the third glucose of 6GXXGGG1, forming 6GXXXGG1 The active sites of the XXTs are “in phase” by virtue of the 51.4° (SI Appendix,Fig.S9). Hence, as long as 6GGXXGG1 is the preferred product in the pool of doubly modified cellohexaoses, helical repeat of the glucan in combination with the 180° offset of the addition of a third xylosyl moiety will never exceed 50%, as the second glucosyl residue of cellobiose (35). In the absence of experimentally observed (33). XXT5, as in plant knockouts, the product would have a GXXG The steric limitation that prevents subsite 2 from accommo- repeat. The absence of steric limitations at subsite 2 of XXT5 dating a xylosyl adduct leads to the N + 2 rule: XXT1 cannot allows xylosyl transfer to glucose N + 2 when glucose N has a transfer a xylosyl residue to the glucosyl residue N + 2 when xylosyl adduct. Therefore, production of a glucan with a consis- glucosyl residue N has a xylosyl adduct. This rule can be ex- tent XXXG repeat, as observed for the native xyloglucan in the tended to XXT2 with some confidence. The homology model of cell wall (11, 29), requires the action of XXT1 and XXT2 to XXT2 (and XXT5) and the crystal structures of XXT1 have synthesize the GXXG repeat, followed by XXT5 to produce nearly identical backbone positions (SI Appendix, Fig. S7A), and XXXG, all of which is organized in a specific manner as to

Fig. 6. Production of an XXXG-type xyloglucan. The model incorporates observed features of the xyloglucans elaborated by the wild-type organism (XXXG repeats) and XXT5-knockout organism (GXXG repeats), the emergence of the reducing end of the glucan from the synthase, the steric requirements imposed by the N + 2 rule (for XXT1 and XXT2), and the lack of a N + 2 rule (for XXT5). Angles in black represent the helical rotation of each cellobiose unit of cellulose, whereas angles in red combine a 180° offset angle with a half-cellobiose helical rotation (25.7°). Circles and stars represent glucose and xylose residues, respectively. The model, based on a 7-glucose separation of XXTs, enables a clear 2D representation. Models based on other separations (11 and 15 glucose units for instance) are possible and may be necessary for allowable protein–protein contacts. This model depicts the order of XXT action required for the XXXG pattern and not the organization of the xyloglucan biosynthetic complex. The XyG biosynthetic complex likely contains multiple copies of all XyG enzymes, producing multiple XyG chains within a single complex (Discussion).

6068 | www.pnas.org/cgi/doi/10.1073/pnas.1801105115 Culbertson et al. Downloaded by guest on September 28, 2021 prevent the xylosylation of all 6-hydroxyl groups of the glucan GGGXXG) for high in vitro activity, the reduction of XXXG- chain (Fig. 6). type xyloglucan in xxt5 knockout plants (29, 30), and why the xxt1/ The linear model shown in Fig. 6 accounts for all that we know xxt2 double knockout has no XyG, whereas the xxt5 single or can be inferred in regard to xylosylation of a glucan chain. knockout retains significant (although reduced) levels of XyG This model does not depict the spatial organization of glucan (29). Experiments to determine the action of XXT5 on specifi- synthase and three XXTs on the 2D surface of the membrane. cally xylosylated substrates and to understand the spatial orga- Information regarding protein–protein interactions is insufficient nization of the XyG biosynthetic complex will lead to a more to favor one packing scheme over another for complex forma- certain understanding of XyG production in plants. The findings tion. The XyG biosynthetic complex likely contains multiple in this study will support strategies to create plant biomass with copies of all XyG biosynthetic proteins and likely synthesizes at desired properties. least two XyG chains within a single complex. In summary, we demonstrate here that XXT1 and/or XXT2 Methods are unlikely to produce the XXXG-type XyG in Arabidopsis, All proteins were expressed in HEK cells as described previously (35). Heavy

requiring the contribution of XXT5 to fully xylosylate the xylo- atom derivative was produced by soaking K2HgI4 in XXT1 crystals. Protein glucan backbone. This limitation of XXT1 and XXT2 and the expression, purification, crystallization, structure determination, and en- function of XXT5 were not apparent from reverse genetics nor zyme assays are described in SI Appendix, SI Materials and Methods. from in vitro studies. Steric constraints revealed by the structures, however, clarify the action of each XXT in xyloglucan ACKNOWLEDGMENTS. We thank Dr. Adam Barb and Dr. Ganesh Subedi for biosynthesis. The ordering of the XXTs proposed here, which sharing the protocols and resources for transformation and growing requires XXT5 to follow XXT1 and XXT2, provides an expla- HEK293 cells, the National Institute of General Medical Sciences, and the Cancer Institutes Collaborative Access Team (Argonne National Laboratory, nation for all previous observations, such as the low activity of Advanced Photon Source; GUP-48455). This study was supported by the XXT5 on nonxylosylated cellohexaose (33), preferring instead a Division of Molecular and Cellular Biosciences, National Science Foundation cellohexaose xylosylated at the N + 2 site (GGGGXG or Grant 1121163 (2011–2017) (to O.A.Z.) and by Roy J. Carver charitable funds.

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