Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of β-1,3-Glucan Quaternary Structure

Article

Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure Graphical Abstract

Authors Benjamin Pluvinage, Alexander Fillo, Patricia Massel, Alisdair B. Boraston

Correspondence [email protected]

In Brief Through the analysis of X-ray crystal structures of the family 81 glycoside hydrolase catalytic module from the Bacillus halodurans protein BH0236 in complex with b-1,3-glucooligosaccharides, Pluvinage et al. reveal the details of this enzyme’s inverting catalytic mechanism and how it likely recognizes b-1,3-glucan quaternary structure.

Highlights d

BhGH81 has a putative endo-processive mode of b-1,3glucan hydrolysis

d

Inverting mechanism with conformational itinerary of 2 S0 / 2,5Bz / 5S1

d

BhGH81 likely recognizes the quaternary structures of helical b-1,3-glucans

Pluvinage et al., 2017, Structure 25, 1–12 September 5, 2017 ª 2017 Elsevier Ltd. http://dx.doi.org/10.1016/j.str.2017.06.019

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Structure

Article Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure Benjamin Pluvinage,1 Alexander Fillo,1 Patricia Massel,1 and Alisdair B. Boraston1,2,* 1Department

of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2017.06.019 2Lead

SUMMARY

Family 81 glycoside hydrolases (GHs), which are known to cleave b-1,3-glucans, are found in archaea, bacteria, eukaryotes, and viruses. Here we examine the structural and functional features of the GH81 catalytic module, BhGH81, from the Bacillus halodurans protein BH0236 to probe the molecular basis of b-1,3-glucan recognition and cleavage. BhGH81 displayed activity on laminarin, curdlan, and pachyman, but not scleroglucan; the enzyme also cleaved b-1,3glucooligosaccharides as small as b-1,3-glucotriose. The crystal structures of BhGH81 in complex with various b-1,3-glucooligosaccharides revealed distorted sugars in the
1 catalytic subsite and an arrangement consistent with an inverting catalytic mechanism having a proposed conformational itinerary of 2S0 / 2,5Bz / 5S1. Notably, the architecture of the catalytic site, location of an adjacent ancillary b-1,3-glucan binding site, and the surface properties of the enzyme indicate the likely ability to recognize the double and/or triple-helical quaternary structures adopted by b-1,3-glucans.

INTRODUCTION As a class, the biological polysaccharides found in, for example, plants, arthropods, and microbes represent a chemically diverse class of macromolecules. They can contain a variety of monosaccharide building blocks, which themselves can be joined via numerous possible distinct glycosidic linkages. An individual monosaccharide in a polysaccharide backbone can also have multiple glycosidically linked substituents, resulting in polysaccharides with varying degrees of branching. Furthermore, depending on the polysaccharide, the monosaccharide building blocks can bear various chemical modifications such as methylesterification, sulfation, and N-acetylation. This diversity manifests as individual species of polysaccharides having distinct structural and physiochemical properties. In nature, such diversity is reflected by the various essentials roles played in living organisms. Some polysaccharides such as starch and glycogen are involved in energy storage; a number

of polysaccharides are involved in building barriers, such as capsules and biofilms, which protect microbes from the environment; other polysaccharides such as agars, pectins, chitin, and cellulose have structural functions. Their biological roles are dependent on the polysaccharide composition, which in turn impart the relevant physiochemical properties, such as tertiary and quaternary structures. For example, cellulose is a component of plant cell walls that helps impart rigidity. This polysaccharide, which is made up of b-1,4-linked glucose, adopts a flat extended ribbon structure where each glucose unit is oriented by a 180 rotation around the glycosidic bond relative to the preceding residue (Beguin and Aubert, 1994). The conformation of cellulose chains results in their self-association into crystalline quaternary structures and, ultimately, cellulose chains arrange into rigid microfibrils that help impart structural integrity to plant cell walls. b-1,3-Glucans are chemically similar polysaccharides but in which the glucose units are b-1,3-linked. These polysaccharides, which can be variably substituted with b-1,6-linked glucose on the b-1,3-linked backbone, are found in plants, fungi, and bacteria in both marine and terrestrial environments, and are therefore relatively abundant. Despite the close chemical relationship of cellulose to b-1,3-glucans, the chains of this latter polysaccharide adopt a helical structure that tend to associate into a triple-helical quaternary structure (Chuah et al., 1983; Deslandes et al., 1980). Thus, homopolymers comprising b-linked glucose that differ only in the substituents through which they are joined have dramatically different structures. Surprisingly, very little is known about the impact of quaternary structure in the degradation of such polysaccharide assemblies. Polysaccharide turnover in biomass is largely performed by the action of microbes that deploy a large group of specialized enzymes known as Carbohydrate Active enZymes (CAZymes) to depolymerize these macromolecules. One of the predominant classes of degradative CAZymes is the glycoside hydrolases (GHs), which are presently grouped into over 130 amino acid sequence-based families and 14 structure- and mechanismbased clans (Cantarel et al., 2009). GHs can act specifically as exo-cleaving enzymes to remove the sugar units from the ends of chains and release small sugar products, or endo-cleaving enzymes to act within the polysaccharide chain and produce oligosaccharides. The enzyme active sites of GHs that accommodate polysaccharide chains are typically described using the subsite nomenclature proposed by Davies et al. (1997). This model defines subsites, each accommodating a monosaccharide unit Structure 25, 1–12, September 5, 2017 ª 2017 Elsevier Ltd. 1

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

and with subsite numbering centered around the scissile bond of the substrate. Plus (+) subsites are consecutively numbered from the point of cleavage toward the reducing end of the polysaccharide while the minus (
) subsites are consecutively numbered from this point to the non-reducing end. Exo-acting enzymes typically have a relatively small number of subsites and usually have only one or two minus or plus subsites that accommodate the non-reducing or, more rarely, reducing end sugars, respectively, that are ultimately released from the polysaccharide by hydrolysis (Davies and Henrissat, 1995). Endo-acting enzymes usually have a larger number of subsites, and the active sites are open at both ends to allow internal binding on polysaccharide chains. The ability of individual active-site subsites to recognize particular monosaccharide substituents of the substrate, and to recognize these when linked in a specific fashion to neighboring monosaccharides, provides GHs with their specificity (Davies and Henrissat, 1995). The efficient hydrolysis of b-1,3-glucans is typically achieved by the activity of both exo-b-1,3-glucosidases (EC 3.2.1.58), which fall into GH families 3, 5, 17, and 55, and endo-b-1,3-glucanases (EC 3.2.1.39) in GH families 16, 17, 55, 64, 81, and 128. The structural analyses of exo-b-1,3-glucanases in complex with b-1,3-glucooligosaccharides elegantly reveal the recognition of the helical tertiary structure adopted by b-1,3-glucan chains. In one case, an ancillary binding site relatively near the catalytic site also binds glucan chains, suggesting more complex recognition of polymerized b-1,3-glucan substrates (Bianchetti et al., 2015). Indeed, surface ancillary polysaccharide binding sites that aid in substrate recognition are not uncommon in GHs (see, e.g., Allouch et al., 2004; Cockburn et al., 2014, 2015, 2016; Ludwiczek et al., 2007); however, in these cases the ancillary binding sites are typically somewhat removed from the catalytic site. Despite the observations that polysaccharide recognition by GHs can be complex, it remains unclear if or how GHs might recognize a quaternary structure in its polysaccharide substrate. In this study we use BH0236, a family 81 glycoside hydrolase from Bacillus halodurans, as a model system to continue to illuminate the molecular details underpinning the recognition and hydrolysis of b-1,3-glucans, particularly the potential to recognize a quaternary structure. Here we use X-ray crystallographic analysis to uncover the features of the catalytic mechanism employed by this enzyme to hydrolyze b-1,3glucans. In doing so, we trapped the structure of a quaternary complex of BhGH81 with two products of catalysis bound in the enzyme active site and a decasaccharide bound in an ancillary site adjacent to the catalytic site, revealing the likelihood that the enzyme is able to recognize a duplex or triplex structure of a b-1,3-glucan. Overall, these results provide new and detailed insight to help guide our understanding of how GHs may recognize non-crystalline polysaccharides that, by virtue of their chemical composition, adopt distinct quaternary structures. RESULTS The b-1,3-Glucanase Activity of BhGH81 on b-1,3Glucans and b-1,3-Glucooligosaccharides The protein BH0236 from B. halodurans is multimodular, comprising a GH81 module at its N terminus, an internal family 6 carbohydrate binding module (CBM), and a predicted family 2 Structure 25, 1–12, September 5, 2017

56 CBM at the C terminus (van Bueren et al., 2005). The boundaries of the catalytic module of BH0236 were predicted by amino acid sequence alignments and the gene fragment encoding this module cloned, overexpressed in Escherichia coli, and purified for further study. This isolated module, referred to as BhGH81, was used to examine the nature of the products released from b-1,3-glucans by its activity using fluorophore-assisted carbohydrate electrophoresis (FACE). The digestion of laminarin for 60 min or overnight resulted in a ladder of products (Figure 1A). The predominant products migrated similarly to the glucose and b-1,3-glucobiose (L2 or laminaribiose) standards while other products had varying degrees of polymerization, presumably ranging to over 6. Digestion of curdlan resulted in the production of products with migration properties that match glucose, L2, and b-1,3-glucotriose (L3 or laminaritriose) (Figure 1B). A 60-min digest of pachyman produced minimal products, but an extended overnight digest resulted in the appearance of products that migrated with the glucose and L2 standards, indicating that BhGH81 does have activity on pachyman (Figure 1B). Extended digestion of insoluble scleroglucan or a gelled fraction of this polysaccharide failed to produce any cleavage products (Figure 1C). A similar analysis using b-1,3-glucooligosaccharides as substrates showed BhGH81 to degrade b-1,3-glucohexaose (L6 or laminarihexaose) and b-1,3-glucopentaose (L5 or laminaripentaose) down to glucose and b-1,3-glucobiose (L2 or laminaribiose) with small amounts of b-1,3-glucotriose (L3 or laminaritriose) (Figure 1D). b-1,3-Glucotetraose (L4 or laminaritetraose) was degraded to L2 and possibly small amounts of glucose while digestion of L3 resulted in incomplete conversion to L2 and glucose. L2 was resistant to enzyme treatment, indicating that L3 is the minimal substrate required for the enzyme (Figure 1E). The time-dependent release of oligosaccharide products from the digestion of laminarin by BhGH81 over 20 min indicated the formation of a wide spectrum of oligosaccharides ranging from glucose up to sugars longer than L6 (Figure 2A). The amount of released oligosaccharides increased but their range of sizes and their relative proportions remained roughly consistent (Figure 2A). After another additional 20 min (40 min in total) the fraction of larger oligosaccharides was reduced while the amount of L3 and L2 increased substantially. It is notable that after extended overnight digestion of laminarin with BhGH81 a group of larger oligosaccharides remains, which indicates a fraction that may be recalcitrant to BhGH81 digestion (Figure 1A). In contrast, a time course of curdlan hydrolysis resulted in the visible production of only L3 up to 20 min; at 40 min L2 was visible as well as potentially a very small amount of L4 (Figure 2B). In an effort to determine kinetic parameters in addition to the time-course product profiling, we also examined the rates of reducing sugar product generation as a function of substrate concentration; however, this resulted in sigmoidal curves and, therefore, Michaelis-Menten parameters could not be determined (Figure S1). We tentatively rationalize the complex kinetics, which may otherwise be interpreted as representing positive cooperativity, as resulting from the use of structurally heterogeneous polysaccharides that potentially present a variety of differentially hydrolyzed substrate conformations (e.g., single glucan chains versus duplex and triplex structures).

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Figure 1. Activity of the BhGH81 Catalytic Module from Bacillus halodurans BH0236 (A–C) FACE analysis of BhGH81 activity on laminarin, curdlan, pachyman, and scleroglucan. The blank in each gel represents a sample lacking both sugar and enzyme and therefore shows the migration of the 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS) label. O/N, overnight. (D and E) FACE analysis of b-1,3-glucooligosaccharide hydrolysis by BhGH81. The substrates are labeled above the gels while the presence or absence of enzyme in the reaction is indicated below the gels. In all panels, L2 to L6 indicate standards ranging from laminaribiose up to laminarihexaose. G indicates the glucose standard. The asterisks indicate bands resulting from the ANTS label.

Structure of BhGH81 and Its Interaction with Products of Catalysis Seleno-methionine substituted (Se-Met) BhGH81 crystallized in the space group P212121, and these crystals were used to solve the structure by a single-wavelength anomalous dispersion experiment that was optimized for the anomalous signal from the incorporated selenium atoms. The completed model of the single protein molecule in the asymmetric unit comprised residues 28–779 of the BhGH81 construct (see Tables S1 and S2 for all diffraction data and model refinement statistics). This catalytic module construct showed a four-domain organization starting from the N terminus of a b-sandwich domain (I, yellow color in Figure 3) that is preceded by a 50-amino-acid sequence with little defined secondary structure (N, orange), an a/b-domain (II, blue), an (a/a)6 barrel domain (III, purple), and finally a small 5-stranded b-sandwich domain (IV, green) (Figure 3). With the exception of the C-terminal domain, this overall structure has been described in detail for the structure of the RmGH81 from Rhizomucor miehei (Zhou et al., 2013), which has a root-mean-square deviation of 2.1 A˚ over 521 matched Cas and 24% amino acid sequence identity with our BhGH81 structure. In RmGH81 the side chains of residues E553 and E557 of RmGH81 were proposed to be the potential acid and base catalytic residues, respectively, with D475 also mentioned in the context of putative catalytic residues. These residues are conserved in BhGH81 as E542, E546, and D466,

which are located in the base of a cleft present near the center of the (a/a)6 barrel (Figure 3). Notably, in the GH81 from Thermobifida fusca (TfGH81), evidence for E499 functioning as the catalytic base, which is conserved with E542 of BhGH81 and E557 of RmGH81, was provided by azide rescue experiments (McGrath et al., 2009). The C-terminal domain of the BhGH81 structure appears to be unique to the B. halodurans enzyme, where it would separate the (a/a)6 barrel domain from the following domain, which is a CBM6 module. However, the RmGH81 structure is lacking 50 amino acids from the C terminus of the crystallized protein, which may comprise another uncharacterized domain similar to that seen in BhGH81. Crystals of BhGH81, which also proved to be in the space group P212121 but with cell dimensions different from those of the Se-Met BhGH81 crystals, were soaked with L6. The resulting structure had a single molecule of BhGH81 in the asymmetric unit with electron density that could be clearly modeled as sugars found in the cleft containing the proposed catalytic residues. Two b-1,3-glucooligosaccharides could be modeled, one molecule of L3 and one of L2, with the non-reducing end of the L2 in close proximity (3.1 A˚) to the reducing end of L3 (Figures 4A and 4B). This arrangement suggests that the two modeled sugars represent cleavage products of L6 with L2 occupying the +1 and +2 subsites and L3 occupying the
1,
2, and
3 subsites. The aromatic side chain of W615 provides the classic CH-p interactions with the sugar unit in the +1 subsite Structure 25, 1–12, September 5, 2017 3

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

tron density consistent with L2 occupying the +1 and +2 subsites and L3 occupying the
1,
2, and
3 subsites (Figure 5A). The interactions in the active-site subsites and the 5S1 conformation of the glucose in the
1 subsite were indistinguishable from the wild-type complex of BhGH81 with the products of L6 hydrolysis (Figure S2). Remarkably, this structure also yielded electron density for a large oligosaccharide that was modeled as b-1,3-glucodecaose (L10 or laminaridecaose) (Figures 5A and 5B). This was found to be bound to the protein in an ancillary binding site immediately adjacent to, and running roughly parallel with, the active site. The most well-defined glucose residues in the L10, as judged by their refined atomic B factors, were the sixth to the second residues from the reducing end and occupied what we defined as ancillary subsites A to E, respectively (Figure 5B). W615, which plays a role in forming the +1 subsite in the catalytic site, interacts with the glucose residue in subsite C, thus resulting in the indole ring of the tryptophan being sandwiched between the pyranose rings of glucose residues bound in the enzyme active site and in this ancillary b-1,3-glucooligosaccharide binding site (Figure 5C). The oligosaccharide adopted a loose helical structure that was large enough to extend over the catalytic cleft and is accommodated by a wide groove running the length of the protein surface (Figure 5D). With the exception of only the reducing end glucose and the two glucose residues over the top of the active-site cleft (the glucose in subsite A and the third residue at the non-reducing end), all of the residues comprising L10 made direct or water-mediated hydrogen bonds with the protein (Figure 5B). Notably, the different packing of protein molecules in the crystal form used to obtain the L6 wild-type product complex blocked this ancillary binding site, preventing its occupation with L6 or its hydrolysis products.

Figure 2. A Product Analysis of Laminarin and Curdlan Hydrolysis Time course of laminarin (A) and curdlan (B) digestion analyzed by FACE. The time points and presence or absence of enzyme in the reaction are indicated below the gels. In all panels, L2 to L6 indicate standards ranging from laminaribiose up to laminarihexaose. G indicates the glucose standard. The asterisks indicate bands resulting from the ANTS label. See also Figure S1.

while W616 does the same for the sugar unit in the
2 subsite. The sugar unit in the
1 subsite was distorted into a 5S1 conformation (Figure 4C) and, notably, this distortion generates a new planar surface comprising carbons 4, 5, and 6 that is not present in the 4C1 chair conformation of glucose, and this new plane packed against the side chain of Y387 (Figure 4B). Stabilization of the distorted conformation of the
1 sugar appeared be further enabled by hydrogen bonds between E542 and O1 and O2 of the sugar, H470 and O6, and E546 and O4. The O3 of the sugar unit in the +1 subsite, which is expected to be positioned roughly where the glycosidic oxygen in the intact substrate would have been, is in position to hydrogen bond with D466. In addition to these notable hydrogen bonds, the interaction between the enzyme active site and sugars is completed by an extensive network of direct and water-mediated hydrogen bonds (Figure 4B). Soaking crystals of BhGH81 (same crystal form as the Se-Metlabeled protein) with laminarin resulted in the observation of elec4 Structure 25, 1–12, September 5, 2017

The Structure of BhGH81 in Complex with an Intact Substrate In an effort to trap a Michaelis-like complex of BhGH81, we sought to inactivate the enzyme by site-directed mutagenesis. On the basis of the proximity of E542 to O1 of the distorted glucose residue in the
1 subsite, we reasoned that this was likely the residue acting as the general catalytic base while the proximity of D466 to O3 of the glucose in the +1 subsite suggested that this was the likely general catalytic acid (Figure 4C). Indeed, substitution of E542 with glutamine or D466 with asparagine resulted in enzymes with undetectable activity on b-1,3glucooligosaccharides as determined by thin-layer chromatography, indicating their importance in catalysis. We proceeded to crystallize BhGH81E542Q and obtained the same crystal form as for the Se-Met labeled protein. When crystals of BhGH81E542Q were soaked with L6, the resulting electron density maps showed two ligands bound in and near the active site (Figure 6A). One molecule of intact (unhydrolyzed) L6 was modeled spanning subsites
3 to +2 of the active site with the final reducing end sugar linked to the sugar residue in the +2 subsite (Figure 6B). The interaction of glucose residues occupying subsites
3,
2, +1, and +2 with the enzyme were identical to those observed in the L6 and laminarin product complexes with wild-type BhGH81. However, the reducing end glucose residue of this L6 molecule made only a single water-mediated hydrogen bond with the backbone carbonyl of W615 and thus did not occupy a clearly definable subsite, and we will therefore

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Figure 3. Structure of BhGH81 Shown from Two Angles The N-terminal segment is colored orange and labeled N. The remaining composite domains I–IV are labeled and colored yellow, blue, purple, and green, respectively. In the right panel the active site is shown as a gray surface with putative catalytic residues colored orange and labeled.

refer to this as the +3* position. The glucose residue in the
1 subsite was distorted into a 2,5B conformation (Figure 5C). As in the unmutated enzyme, Q542 (E542 in the wild-type) maintained a hydrogen bond with O2 of the glucose in the
1 subsite while a carboxylate oxygen of D466 was within 3 A˚ of the glycosidic oxygen between the glucose residues in
1 and +1 subsites. However, because of the boat conformation adopted by the sugar in the
1 subsite, Q542 interacted with a water molecule that sits 3.3 A˚ beneath C1 (Figure 6C). As with the laminarin product complex, we observed electron density for a second oligosaccharide molecule, which was modeled as L5, bound in the ancillary site and occupying subsites A to E (Figure 6B). In general, the same series of interactions were evident between the enzyme and L5 in the ancillary site as for the L10 in the laminarin complex. However, also evident in this complex was a water network bridging the glucose residues of L5 in ancillary subsites B, C, and D, with the two glucose residues at the reducing end of the L6 molecule occupying the catalytic site, likely resulting in the ordered conformation of the reducing end glucose of L6 at the +3* position (Figure 6B). Structural Mapping of Subsites in the Catalytic and Ancillary Sites In addition to determining the structure of BhGH81E542Q in complex with L6, to probe the occupation of individual subsites we also determined the structure of this mutant in complex with L2, L3, L4, and L5. Like with L6, the electron density for L5 was clear and continuous and terminated cleanly at O1 of the reducing end and O3 of the non-reducing end, indicating only a single poise for the sugar (Figure 7). The refined conformation of L5 indicated the same Michaelis-like complex observed for L6, with subsites
3 to +2 occupied. In contrast, the electron density observed in the L4 complex was somewhat ambiguous (Figure 7). Overall, the electron density for an oligosaccharide appeared to be continuous and span five subsites (
3 to +2), despite using a ligand with only four monosaccharide units. Consistent with the presence of an intact glycosidic bond spanning the catalytic machinery, the conformation of the glucose residue in the
1 subsite could

be modeled in the 2,5B conformation observed in the Michaelis-like complexes (i.e., L6 and L5 complexes). However, unlike the Michaelis-like complexes, a distinct water molecule was not evident immediately below C1 of the glucose in the
1 subsite. Rather, the electron density for C1 was continuous with an atom pseudo-axial to this position. Notably, the L4 structure was determined at a resolution similar to that of the L6 and L5 complexes and had very similar quality indicators. This is evidence of glucose in the
1 subsite with an alternative conformation, specifically, the same 5 S1 conformation observed for the reducing end sugar in the product complexes (Figures 4 and 5). Thus, to account for this latter observation the alternative conformation of L4 was modeled as mimicking a product complex with L3 occupying subsites
1 to
3 and the fourth non-reducing end glucose presumably being disordered (Figure 7). The other conformation, which spanned the catalytic machinery, was modeled as a Michaelis-like complex of L4 occupying subsites
2 to +2. The electron density terminated cleanly at O1 of the glucose in the +2 subsite, indicating insignificant occupation of the
1 to +3* subsites by a molecule of L4; however, given the data, we cannot discount the possibility of the presence of L4 also positioned in the
3 to +1 subsites. The L3 complex revealed electron density for two distinct L3 molecules, one modeled occupying the +1 to +3* subsites and the other occupying the
3 to
1 subsites with the terminal glucose in the
1 subsite in a 5S1 conformation (Figure 7). Two clearly defined sugar molecules in the L2 complex were also observed, with L2 occupying the +1 to +2 subsites and another occupying the
3 to
2 subsites (Figure 7). To ensure that the lack of occupancy of the
1 subsite in the L2 complex was not a result of the E542Q mutation, we also determined the structure of the unmutated BhGH81 with L2 and observed the same binding mode (Figure 7). Like with L5, the electron density for the sugars modeled in the L2 and L3 complexes terminated cleanly at their reducing and non-reducing ends, indicating no evidence of multiple binding modes. Overall, for all of the L2 to L5 complexes, the specific interactions between the sugar units and the subsites in the active site were the same as those observed in the complex of BhGH81E542Q with L6. The pattern of subsite occupancy in the ancillary binding site was less complicated than the catalytic site occupancy. L5 occupied all five subsites, A to E, as was found in the L6 complex, and L4 clearly occupied sites B to E (Figure 7). The L3 complex also showed electron density in subsites C and D that was modeled as L2 (Figure 7). The density was clearly that resulting Structure 25, 1–12, September 5, 2017 5

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Figure 4. Structure of BhGH81 in Complex with Reaction Products from L6 Cleavage (A) Electron density for oligosaccharides bound to BhGH81 in the L6 product complex. The blue mesh shows the electron density for these sugars as a sa-weighted Fo-Fc omit map contoured at 3s. The L3 product is shown as blue sticks and the L2 product as orange sticks. Active-site subsites are indicated in red. (B) The active site in complex with products resulting from L6 cleavage. L3 is shown as blue sticks and L2 as orange sticks. Relevant amino acid side chains are shown as gray sticks, waters as red spheres, and putative hydrogen bonds as gray dashes. The side chain proposed to act as the acid, D466, is colored magenta and the proposed base, E542, is colored blue. Active-site subsites are labeled in red. (C) A close-up of the catalytic machinery and glucose residue occupying the
1 subsite, which shows its distortion to a 5S1 conformation. The blue mesh shows the electron density for this glucose residue as a sa-weighted Fo-Fc omit map contoured at 3s.

from a sugar; however, it was only clear at comparatively low contouring of the electron density (0.5s, 0.22 e
/A˚3) and it was only partial, thus indicating quite low occupancy. In contrast, in the L2 complexes no electron density that was clearly interpretable as sugar was observed in the ancillary binding site (Figure 7). DISCUSSION Catalytic Mechanism Through nuclear magnetic resonance studies of TfGH81, the general catalytic mechanism used by family 81 GHs was determined to be an inverting mechanism (McGrath and Wilson, 2006). Subsequent functional studies, primarily of the GH81 enzymes from Saccharomyces cerevisiae, RmGH81, and TfGH81, have pointed to three amino acid residues that are conserved in the family as candidate catalytic residues: an aspartate (D466 in BhGH81) and two glutamates (E542 and E546 in BhGH81) (Martin-Cuadrado et al., 2008; McGrath et al., 2009; Zhou et al., 2013). While the positions of this trio of residues in the RhGH81 structure are generally consistent with roles in catalysis the most convincing data remain the azide rescue of the 6 Structure 25, 1–12, September 5, 2017

TfGH81E499A mutant, which indicated E499A (E542 in BH0236) as being the catalytic base. Our structures in complex with products and unhydrolyzed substrate generally support these existing reports while illuminating the molecular details of the GH81 catalytic mechanism. The product complex of BhGH81 revealed the L3 product to have the reducing end glucose in the
1 subsite and in a 5S1 conformation. This conformation was also observed for the reducing end glucose unit in the
1 subsite of the laminarin complex. The glucose unit in the
1 subsite of the unhydrolyzed L6 trapped on the BhGH81E542Q mutant, however, was in a 2,5B conformation. These results imply a conformational itinerary of 2S0 / 2,5Bz / 5S1, where the unhydrolyzed substrate complex we obtained more closely resembles the predicted transition state (2,5Bz) than the anticipated Michaelis complex (2S0). This ‘‘classical’’ conformational itinerary is proposed to be utilized by members of GH families 6, 8, 11, 39, 78, and 120, with both retaining and inverting catalytic mechanisms represented by these families (Speciale et al., 2014). Support for the catalytic mechanism of BhGH81 proceeding with inversion of the stereochemistry at the anomeric carbon is observed in the arrangement of the catalytic residues. One of the trio of conserved predicted catalytic residues, E546, is not positioned appropriately to participate directly in catalysis. Rather, in both the product complex and unhydrolyzed substrate complex, E546 hydrogen bonds with the pseudo-axial O4 of the distorted sugar in the
1 subsite, therefore likely assisting in stabilizing the altered conformation of the sugar. Consistent with this, alanine substitution of the analogous residue in TfGH81, E503, resulted in a dramatic reduction in activity (McGrath et al., 2009). In the product complex the carboxylate of D466

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Figure 5. Structure of BhGH81 in Complex with Reaction Products from Laminarin Cleavage (A) The blue mesh shows the electron density for the glucose residues as a sa-weighted Fo-Fc omit map contoured at 3s. The oligosaccharide bound in the ancillary site is shown as green sticks and the oligosaccharides bound in the active site as orange and blue sticks. Active-site subsites are indicated in red numbers and ancillary binding subsites in red uppercase letters. (B) Two views of the product complex of BhGH81 obtained by soaking crystals in laminarin focusing on the L10 molecule occupying the ancillary binding site near the catalytic site. The additional L3 and L2 products of hydrolysis are shown as transparent blue and orange sticks, respectively (see also Figure S2). The ancillary binding subsites are labeled as red uppercase letters. (C) A close-up of W615 in the laminarin complex showing how it is sandwiched between glucose residues in the +1 and +2 catalytic sites and glucose residues in the L10 molecule bound in the ancillary binding site. The ancillary binding subsites are labeled as red uppercase letters. (D) The solvent-exposed surface of BhGH81 in complex with the products of laminarin hydrolysis and colored by electrostatic potential. The L10 molecule is shown as green sticks, the L3 as blue sticks, and the L2 as orange sticks.

Structure 25, 1–12, September 5, 2017 7

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Figure 6. Structure of BhGH81E542Q in Complex with L6 (A) The blue mesh shows the electron density for this glucose residue as a sa-weighted Fo-Fc omit map contoured at 3s. The oligosaccharide bound in the ancillary site is shown as green sticks and the oligosaccharide bound in the active site as orange sticks. Active-site subsites are indicated in red, and ancillary binding subsites in red uppercase letters. (B) The active site in complex with two molecules of non-hydrolyzed b-1,3-glucooligosaccharides. L5 was modeled into the ancillary binding site and is shown as green sticks. The L6 in the active site is shown as orange sticks. The W615 side chains separating the ancillary site and active site is shown as gray sticks. The proposed acid catalyst, D466, is colored magenta and the position of the proposed base, E542, is colored blue. Waters bridging the oligosaccharides in the ancillary binding site and active site are shown as red spheres, and putative hydrogen bonds as gray dashes. The ancillary binding subsites are labeled as red uppercase letters. (C) A close-up of the catalytic machinery and glucose residue occupying the
1 subsite, which shows its distortion to a 2,5B conformation. The blue mesh shows the electron density for this glucose residue as a sa-weighted Fo-Fc omit map contoured at 3s. The catalytic residues are shown as sticks and the putative catalytic water is shown as a red sphere.

hydrogen bonds with O3 of the leaving group sugar, while in the unhydrolyzed substrate complex this residue is ideally positioned to donate a proton to the glycosidic oxygen involved in the scissile bond. Furthermore, our D466N mutation had qualitatively little to no activity while the alanine substitution of the analogous residue in TfGH81, D422, also had a dramatic reduction in activity. These observations are consistent with the assignment of D466 as the acid catalyst. The predicted catalytic base in BhGH81 is E542, which to obtain the L6 substrate complex was mutated to glutamine. In the L6 substrate complex the side-chain amide of Q542 is positioned 3.7 A˚ below C1 of the distorted glucose in the
1 subsite but displaced 1.5 A˚ to the side such that the amide nitrogen may hydrogen bond with O2 (Figure 6C). A water molecule that is positioned 3.1 A˚ from the Q542 side-chain amide nitrogen with appropriate geometry to hydrogen bond sits 3.1 A˚ beneath C1 and O5. Given that Q542, which would otherwise be a glutamate, is not well positioned to directly attack C1, this arrangement suggests that the water molecule, which is reasonably well positioned to attack beneath C1, is a catalytic water that is activated by the E542 8 Structure 25, 1–12, September 5, 2017

catalytic base. Supporting this, in the product complex one of the side-chain carboxylate oxygens of E542 hydrogen bonds with both O2 and O1 (both 2.7-A˚ bonds) of the glucose in the
1 subsite where O1 is now in an axial position and occupies roughly the same space as the proposed catalytic water. In summary, therefore, the features of the BhGH81 active site are coherent with an inverting catalytic mechanism of b-1,3-glucan hydrolysis where the side chains of E542 and D466 act as the catalytic base and acid, respectively. Subsite Occupation The tendency of BhGH81 to generate a pool of oligosaccharides with larger degrees of polymerization in the initial stages of laminarin digestion, but which are subsequently degraded as digestion proceeds, is most consistent with an apparent endo mode of chain cleavage (Chi et al., 2014; Labourel et al., 2015). However, it is notable that the enzyme also produced L3 as the major product from the initial stages of laminarin digestion, which gives the impression of a degree of exo-b-1,3-glucanase activity. To provide some insight into this, we probed the sequential occupation of the catalytic subsites through structural analysis of complexes of BhGH81E542Q using a series of b-1,3-glucooligosaccharides. BhGH81 displayed no detectable activity on L2, which was consistent with the formation of the non-productive complexes

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Figure 7. Schematic Representing Occupation of the Active-Site Subsites and the Ancillary Binding Sites in the BhGH81E542Q Mutant by b-1,3-Glucooligosaccharides The normal point of glycosidic bond cleavage is shown by the orange arrows. The red circles indicate glucose residues in the 2,5B conformation of the Michaelis-like complex while the blue circles indicate the 5S1 glucose conformation observed in the product complexes. NR and R refer to the nonreducing and reducing ends of the sugars, respectively. The sugar marked ‘‘a’’ was only partially occupied. +3* indicates an ordered glucose residue that does not occupy a clearly defined subsite. N/A indicates no occupation of these binding sites was observed. See also Figures S3–S6.

of L2 in with the wild-type and mutant enzymes where the
3 and
2 subsites accommodated an L2 and the +1 and +2 subsites bound another. These results suggest that the
3,
2, +1, and +2 subsites possess the highest affinity for glucose residues. The complex with L3 also revealed a non-productive complex having two L3 molecules bound in the active site with neither spanning the catalytic machinery. This contradicts the activity data, which revealed the ability of BhGH81 to incompletely convert L3 into L2 and glucose, thus indicating that L3 is able to form a catalytically productive complex with the enzyme by occupying the
2 to +1 subsites or the
1 to +2 subsites. In the complexed structure, L4 formed both a non-productive complex, with only the
3 to
1 subsites occupied, and a complex spanning the catalytic residues including the
2 to +2 subsites. An alternative complex spanning the catalytic residues that may be formed but which would not be directly observable in the electron density would be one spanning the
3 to +1 subsites. However, the activity data of BhGH81 on L4 revealed only the production of L2; the amounts of glucose present in this sample were similar to those seen in all of the samples in the absence of added enzyme. Thus, the catalytically productive binding mode of L4 is most likely one that spans
2 to +2. This is also consistent with the preferential occupation of the +1 and +2 subsites by L2 alone and leads to the suggestion that the catalytically productive binding mode of L3 involves the
1 to +2 subsites, thus resulting in the release of the reducing end b-1,3-glucobiose from L3. The activity of BhGH81 on L5 resulted in predominantly L2 and glucose with small amounts of L3. The observed asymmetric binding mode of L5 in the complex, whereby subsites
3 to +2 were occupied, is consistent with this and points to an initial cleavage releasing L2 from the +1 and +2 subsites and subsequent cleavage of the remaining L3 into L2 and glucose. Like with the activity of the enzyme on L5,

it hydrolyzed L6 into L2 and glucose with small amounts of L3. The symmetric mode of L6 binding in the substrate complex with this sugar indicates that this pattern of product generation likely results from the initial cleavage of L6 into two molecules of L3 and subsequent cleavage of the L3 into L2 and glucose. Thus, L2 generated from laminarin hydrolysis likely results primarily from the hydrolysis of L3, L4, and L5, which is consistent with production of L2 at later time points in laminarin depolymerization. The observed mode of L6 binding, whereby it is symmetrically bound in the catalytic site with no obvious impediment to additional glucose residues on both the reducing and non-reducing ends of the molecule, supports the capacity of BhGH81 to cleave laminarin with apparent endo-hydrolytic activity. The specific pattern of subsite occupation, however, indicates preferential occupation of the
3,
2, +1, and +2 subsites. On this basis, we surmise that the production of L3 observed early in laminarin hydrolysis, prior to the build-up of significant amounts of oligosaccharide fragments that would be further degraded, may result from an ability of the enzyme to also bind and cleave at glucan chain ends. The lack of three distinct subsites to bind the reducing end of the polymer points makes binding the nonreducing end most likely where the
1 to
3 subsites are occupied by three terminal glucose residues and hydrolysis would result in the release of L3. b-1,3-Glucan Quaternary Structure and a Model of Substrate Recognition and Cleavage The identification of the ancillary glucan binding site in the BhGH81 active site was unexpected but significant in light of the structure adopted by b-1,3-glucans. b-1,3-Glucan chains have a tendency to form helices, which in turn have a propensity to associate into a quaternary structure comprising a triple helix of parallel chains; imperfections in the association of individual chains can lead to disordered loop regions or the formation of duplexes (Bacic et al., 2009). Our complexes of BhGH81E542Q with b-1,3-glucooligosaccharide and the unmutated enzyme with laminarin fragments clearly reveal discrete oligosaccharides bound to the ancillary binding site and the catalytic site. These oligosaccharides run parallel to one another and even interact via a water network, indicating that the ancillary site and catalytic site cannot be simultaneously occupied by a single contiguous b-1,3-glucan chain. Therefore, these structures indicate that the enzyme must be able to bind at least two separate b-1,3glucan chains simultaneously, implying the potential to recognize duplex or triplex structures. To build upon this concept, we used the helical structure of L10 observed in the laminarin complex as a template to superimpose the triple-helical structure determined for curdlan (Chuah et al., 1983). This suggested that the cylindrical shape of a b-1,3-glucan triple helix, and therefore single and double helices, is not only well mirrored by the contours of the enzyme surface in the immediate vicinity of the ancillary binding site and catalytic site but is also mirrored across the full dimension of the enzyme (Figure 8A). More specifically, the structure of L10 and the arrangement of the two product oligosaccharides, L2 and L3, roughly mimic the overall helical structure of two separate chains in the triple helix model (Figure 8B). However, agreement between the positions of the sugars in the experimental structures and the positions of the chains in the modeled triple helix are Structure 25, 1–12, September 5, 2017 9

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Figure 8. Comparison of the Curdlan Triple-Helical Structure with the Surface Properties of BhGH81 (A) The solvent accessible surface of BhGH81 (gray) showing the bound L10 (green), L2 (purple), and L3 (purple) molecules from the laminarin product complex. The triple-helical structural determined for curdlan II is shown as orange, yellow, and blue b-1,3-glucan chains (Chuah et al., 1983). The curdlan triple helix was placed by overlapping the yellow b-1,3-glucan chain in the curdlan structure with the L10 molecule in the BhGH81 complex. The pitch of the helix formed by a single glucan chain in curdlan is 16 A˚ while the separation between adjacent chains in the triple helix is 5.3 A˚. (B) A close-up of the BhGH81active site showing that the oligosaccharides engaging the enzyme’s catalytic machinery are displaced down into the active site 6–7 A˚ relative to the chains in the modeled curdlan triple helix.

sufficiently different to warrant consideration. First, the separation between individual chains in the curdlan triple helix is 5.3 A˚ (Figure 8A), whereas the ridge in the enzyme between the active site and ancillary binding site impose a distance of 7.0 A˚ between pairs of bound b-1,3-glucan chains (Figure 8B). Second, the helix adopted by L10 has a larger pitch than that of the helices in curdlan (16.9 A˚ and 8 glucose residues per turn versus 16.0 A˚ and 7 glucose residues per turn in an ideal curdlan helix) and is thus a slightly more open helix. Lastly, the sugars present in the catalytic site of the enzyme are deep in the catalytic cleft and, relative to the chains of the modeled curdlan triple helix, 10 Structure 25, 1–12, September 5, 2017

are displaced away from the central axis of the curdlan triple helix by approximately 6–7 A˚ (Figure 8B). Therefore, the architectures of the ancillary binding site and catalytic sites considered together argue for the recognition of a b-1,3-glucan quaternary structure with more open and/or relaxed helical structures than is present in the glucan chains of the triple helix. With respect to this, a primary consideration is that the ridge intervening between the ancillary binding site and the catalytic site necessitates a distance of 7 A˚ between parallel glucan chains. This might be accomplished if the recognition of the triple helix by BhGH81 resulted in a local twisting of the triple helix to open it slightly and achieve separation between the glucan chains, thus promoting binding and access of a glucan chain to the catalytic site. This is formally possible but implies a potentially large energetic cost. An alternative is that BhGH81 recognizes duplex regions of b-1,3-glucans where the separation between the two chains forming the duplex could be up to 11 A˚, which would be sufficient to accommodate the wall separating the ancillary binding site and the catalytic site while allowing access of one of the chains to the catalytic site without the need for gross distortions to the double helix of the polysaccharide. These hypotheses regarding b-1,3-glucan quaternary structure recognition presume an endo-hydrolytic mode of polysaccharide chain binding and cleavage. While the activity data on laminarin is most conservatively interpreted as showing an endo mode of chain cleavage, we note some additional contradictory observations that require consideration. The hydrolysis profiles of curdlan and pachyman, which are non-branched b-1,3-glucans, revealed the production of only small soluble products of primarily glucose, L2, and L3 (Figure 1B); indeed, this was notably the case for the early time points in curdlan hydrolysis, which produced mainly L3 (Figure 2B). This is not consistent with an endo mode of polysaccharide cleavage, but is what would be expected for an exo-hydrolytic mode of activity. In contrast, as we already noted, the product profile at the initial time points of laminarin hydrolysis giving the appearance of a somewhat mixed endo-/exo-hydrolytic cleavage. The structural data clearly reveal the capability of the enzyme’s catalytic site to accommodate internal binding on glucan chains, consistent with an endo mode of action. However, simultaneous binding of the catalytic site and the adjacent ancillary binding site to intertwined b-1,3-glucan chains would result in avid binding to the substrate, and thus an endo mode of activity would require both the catalytic site and ancillary site to completely release their bound glucan chains for the enzyme to reposition for additional cleavage events. As this would likely be energetically and kinetically limiting, it is difficult to reconcile a purely endo mode of activity with the structurally observed mechanism of substrate recognition and some of the observed activity results. On this basis, we offer an additional hypothesis that BhGH81 acts on b-1,3glucan duplex or triplex helical structures in an endo-processive manner. That is, the enzyme makes an initial endo-cut in one chain of the helical substrate, resulting in a new non-reducing end that remains bound to the active site in the +1 to +3* subsites. By slipping along this newly liberated non-reducing chain end, which would likely require no large distortions of the overall glucan quaternary structure other than keeping the free chain end pulled away from the substrate’s helical organization, glucose residues could then occupy the
1 and
3 subsites,

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followed by bond hydrolysis. Repetition of this process, driven by the favorability of the non-reducing end glucose residues to occupy the minus (
) subsites, would result in processive cleavage from the non-reducing end of the glucan chain to systematically produce L3, as was observed for the time course of curdlan hydrolysis. Accommodation of the overall helical quaternary structure of the b-1,3-glucan and the potential for the chain to slide through the ancillary binding site, but not be released, might be envisioned to promote processive activity. Similarly, Bianchetti et al. (2015) proposed a processive exo-hydrolytic mode of laminarin cleavage for SacteLam55 from Streptomyces sp. SirexAA-E. This latter hypothesis presumes a double- or triple-helical b-1,3-glucan that lacks b-1,6-branches, such as curdlan and pachyman (Bacic et al., 2009), where there would be no impediments to processive activity. Indeed, the architecture of the BhGH81 catalytic site indicates that the enzyme is likely incapable of accommodating b-1,6-substitutions on glucose residues occupying the
3 to +1 subsites, as the substitutions would clash with the enzyme surface. Likewise, the ancillary binding site has features consistent with an inability to tolerate b-1,6-substitutions on glucose residues in the B, C, and D subsites. These particular structural impositions likely explain why BhGH81 possesses no activity on scleroglucan, which is highly substituted with one b-1,6-branch every three residues of the glucan backbone (Bacic et al., 2009). The structure of laminarin likely also contributes to the somewhat ambiguous activity results obtained with this polysaccharide. Laminarin has an average degree of polymerization of 25 with a b-1,6-glucose branch every 10 backbone residues (on average) and contains only 5% triple-helical structure (Oda et al., 2016). With respect to our model of activity, the low content of triple-helical structure would be predicted to influence the processive action of the enzyme, as simultaneous occupation of the ancillary binding site and catalytic site by associated glucan chains would be comparatively rare. Furthermore, although the b-1,6-glucose substitutions are likely not frequent enough to prohibit an initial endo-cleavage, they would present obstructions and limit the degree of processivity as well as result in fragments that are ultimately recalcitrant to hydrolysis, which was observed. Thus, although the precise details of the steps between recognition of the b-1,3-glucan quaternary structure and glycosidic bond hydrolysis remain a matter of speculation, we presently favor the hypothesis that BhGH81 acts via an endo-processive mechanism, as this is the most readily rationalized given the present data. We do, however, acknowledge that the construct used in the present study lacks the ancillary modules of the complete BH0236 protein, and the potential role of these ancillary modules in b-1,3-glucan hydrolysis is unknown. Indeed, our proposed mode of endo-processive action from the non-reducing end of glucan chains would likely be promoted by an initial endo-cleavage close to a non-reducing end, and this targeting could be performed by the internal CBM6 module that binds the non-reducing ends of b-1,3-glucan chains (van Bueren et al., 2005). An appreciated characteristic of polysaccharides is their energetically favorable adoption of specific tertiary structures, which is frequently observed to play a role in the molecular recognition of polysaccharides by proteins. However, the adoption of qua-

ternary structures by polysaccharides is less frequently considered, particularly with respect to the concept of molecular recognition by proteins, and is typically only thought of in the context of crystalline polysaccharides. Our structural observations support the general hypothesis that BhGH81 has the capacity to recognize the double- and/or triple-helical quaternary structure formed by b-1,3-glucans and builds our understanding of the molecular requirements for the recognition of non-crystalline polysaccharides that, by virtue of their chemical composition, adopt distinct quaternary structures. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING METHOD DETAILS B Materials B Cloning B Protein Expression and Purification B Enzyme Activity Assays B Enzyme Kinetic Assays B General Crystallography Procedures B BhGH81 Structure Determination DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.str.2017.06.019. AUTHOR CONTRIBUTIONS Conceptualization, A.B.B.; Methodology, A.B.B., B.P., P.M., and A.F.; Investigation, B.P., P.M., and A.F.; Writing – Original Draft, A.B.B., B.P., and A.F.; Writing – Review & Editing, A.B.B. and B.P.; Funding Acquisition, A.B.B.; Supervision, A.B.B. ACKNOWLEDGMENTS This research was supported by a Natural Sciences and Engineering Research Council of Canada Discovery grant to A.B.B. (FRN 04355). We thank the staff at the Canadian Light Source (CLS) where diffraction data were collected. We also thank the beamline staff at the Stanford Synchrotron Research Laboratory BL11-1. Received: April 12, 2017 Revised: June 13, 2017 Accepted: June 30, 2017 Published: August 3, 2017 REFERENCES Abbott, D.W., Higgins, M.A., Hyrnuik, S., Pluvinage, B., Lammerts van Bueren, A., and Boraston, A.B. (2010). The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae. Mol. Microbiol. 77, 183–199. Allouch, J., Helbert, W., Henrissat, B., and Czjzek, M. (2004). Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose. Structure 12, 623–632. Bacic, A., Fincher, G.B., and Stone, B.A. (2009). Chemistry, Biochemistry and Biology of (1-3)-Beta-Glucans and Related Polysaccharides (Academic Press).

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McKee, L.S. (2017). Measuring enzyme kinetics of glycoside hydrolases using the 3,5-dinitrosalicylic acid assay. In Protein-Carbohydrate Interactions Methods and Protocols, D.W. Abbott and A. Lammerts van Bueren, eds. (Humana Press), pp. 27–36. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255. Oda, M., Tanabe, Y., Noda, M., Inaba, S., Krayukhina, E., Fukada, H., and Uchiyama, S. (2016). Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses. Carbohydr. Res. 431, 33–38. Pluvinage, B., Higgins, M.A., Abbott, D.W., Robb, C., Dalia, A.B., Deng, L., Weiser, J.N., Parsons, T.B., Fairbanks, A.J., Vocadlo, D.J., et al. (2011). Inhibition of the pneumococcal virulence factor StrH and molecular insights into N-Glycan recognition and hydrolysis. Structure 19, 1603–1614. Powell, H.R. (1999). The Rossmann Fourier autoindexing algorithm in MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 55, 1690–1695. Speciale, G., Thompson, A.J., Davies, G.J., and Williams, S.J. (2014). Dissecting conformational contributions to glycosidase catalysis and inhibition. Curr. Opin. Struct. Biol. 28, 1–13. van Bueren, A.L., Morland, C., Gilbert, H.J., and Boraston, A.B. (2005). Family 6 carbohydrate binding modules recognize the non-reducing end of beta-1,3-linked glucans by presenting a unique ligand binding surface. J. Biol. Chem. 280, 530–537. Vonrhein, C., Blanc, E., Roversi, P., and Bricogne, G. (2007). Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230. Zhou, P., Chen, Z., Yan, Q., Yang, S., Hilgenfeld, R., and Jiang, Z. (2013). The structure of a glycoside hydrolase family 81 endo-beta-1,3-glucanase. Acta Crystallogr. D Biol. Crystallogr. 69, 2027–2038.

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

ATCC

ATCC BAA-125D

Bacterial and Virus Strains Bacillus halodurans Chemicals, Peptides, and Recombinant Proteins Laminarin

Sigma-Aldrich

Cat#L9634

Laminarihexaose

Megazyme

Cat#O-LAM6

Laminaripentaose

Megazyme

Cat#O-LAM5

Laminaritetraose

Megazyme

Cat#O-LAM4

Laminaritriose

Megazyme

Cat#O-LAM3

Laminaribiose

Megazyme

Cat#O-LAM2

Curdlan

Megazyme

Cat#P-CURDL

Pachyman

Megazyme

Cat#P-PACHY

Scleroglucan

V-labs

Cat#PS135

8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt

Sigma-Aldrich

Cat#08658

Cyanoborohydride

Sigma-Aldrich

Cat#156159

SelenoMethionine Medium Complete

Molecular Dimensions

Cat#MD12-500

Deposited Data BhGH81 seleno-methionine structure

This paper

PDB: 5T49

BhGH81 structure in complex with laminarin

This paper

PDB: 5T4G

BhGH81 structure in complex with L2

This paper

PDB: 5V1W

BhGH81 structure in complex with L6

This paper

PDB: 5T4A

BhGH81E542Q structure in complex with L2

This paper

PDB: 5UPI

BhGH81E542Q structure in complex with L3

This paper

PDB: 5UPM

BhGH81E542Q structure in complex with L4

This paper

PDB: 5UPN

BhGH81E542Q structure in complex with L5

This paper

PDB: 5UPO

BhGH81E542Q structure in complex with L6

This paper

PDB: 5T4C

Invitrogen

Cat#C601003

Primer GH81_Forward: 5’-GGCGGATCTAGACATGCGGTGAGCGTCGGG-3’

This paper

N/A

Primer GH81_Reverse: 5’-TCCGCCCTCGAGTTACGGCTCTGAAGGATCTGG-3’

This paper

N/A

Primer GH81E542Q_Forward: 5’-GGGAACAATCAGCAATCTTCCTCGGAAGCG-3’

This paper

N/A

Primer GH81E542Q_Reverse: 5’-CGCTTCCGAGGAAGATTGCTGATTGTTCCC-3’

This paper

N/A

Primer GH81D466N_Forward: 5’-ACGCGAATTAATAACCATCATTTCCATTAC-3’

This paper

N/A

Primer GH81D466N_Reverse: 5’-GTAATGGAAATGATGGTTATTAATTCGCGT-3’

This paper

N/A

Novagen/Millipore

Cat#69864

CCP4 suite

Collaborative Computational Project, Number 4, 1994

http://www.ccp4.ac.uk

Coot

Emsley and Cowtan, 2004

http://www.ccp4.ac.uk

Experimental Models: Organisms/Strains Escherichia coli BL21 Star (DE3) Oligonucleotides

Recombinant DNA Plasmid: pET28a (+) DNA Software and Algorithms

(Continued on next page)

Structure 25, 1–12.e1–e3, September 5, 2017 e1

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Molprobity

Davis et al., 2007

https://www.phenix-online.org

autoSHARP

Vonrhein et al., 2007

http://www.embl-hamburg.de/ARP/

ARP/wARP

Langer et al., 2008

http://www.ccp4.ac.uk

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Alisdair B. Boraston ([email protected]). METHOD DETAILS Materials b-1,3-glucooligosaccharides, curdlan and pachyman were obtained from Megazyme International Ireland Ltd. (Bray Co., Wicklow, Ireland). Scleroglucan was obtained from V-Labs (Covington, LA). All reagents, chemicals and other carbohydrates were purchased from Sigma unless otherwise specified. Cloning Gene fragments encoding the catalytic module BhGH81 (amino acids boundaries 29-790) was amplified by PCR from B. halodurans genomic DNA (ATCC BAA-125D) and cloned into pET28a via the engineered restriction sites using standard molecular biology procedures. Site-directed mutations E542Q and D466N were introduced using the ‘‘megaprimer’’ PCR method (Barik, 1996). The oligonucleotide primers used for PCR are given in the Key Resources Table. The resulting gene fusions encoded a N-terminal six-histidine tag fused to the protein of interest by an intervening thrombin protease cleavage site. Bidirectional DNA sequencing was used to verify the fidelity of each construct. Protein Expression and Purification All recombinant expression vectors were transformed into E. coli BL21 Star (DE3) cells (Invitrogen) and proteins were produced using 2xYT medium supplemented with kanamycin (50 mg/ml). Briefly, bacterial cells transformed with the appropriate expression plasmid were grown at 37 C until the culture reached an optical density of 0.9 at 600 nm. Gene expression and protein production was then induced by the addition of isopropyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM; followed by overnight incubation at 16 C with shaking. Cells were harvested by centrifugation and disrupted by chemical lysis (Pluvinage et al., 2011). Proteins were purified from the cleared cell-lysate by Ni2+-immobilized metal affinity chromatography followed by size exclusion chromatography using a Sephacryl S-100 or S-300 column (GE Healthcare). Purified protein was concentrated using a stirred-cell ultrafiltration device with a 5 or 10 K molecular weight cut-off membrane (Millipore). Selenomethionine-labeled BhGH81 was produced using E. coli BL21 Star (DE3) in 4L of SelenoMet Medium Base (Molecular Dimensions Ltd.) supplemented with SelenoMet Nutrient Mix (Molecular Dimensions Ltd.) and L-selenomethionine (40 mg/L). These cultures were grown, induced, harvested; and the protein was purified and concentrated using the same method as for unlabeled protein. Protein concentration was determined by measuring the absorbance at 280 nm and using the calculated molar extinction coefficient of 189,540 cm-1 x M-1 for BhGH81 and BhGH81E542Q (Gasteiger et al., 2003). Enzyme Activity Assays Reaction products were analyzed by Fluorophore-assisted carbohydrate electrophoresis (FACE) in a protocol adapted from Abbott et al. (Abbott et al., 2010). Briefly, 0.5 mM of enzyme was incubated overnight in presence of 5 mg/ml of b-1,3-glucan or b-1,3-glucooligosaccharides in 10 ml reaction volume in Tris 20 mM pH 8.0. The reactions were stopped by addition of 1 ml of ice cold 100% ethanol and the samples dried using a vacuum concentrator at 50 C for 2 hrs. Overnight labeling of the sugar products was carried out at 37 C by adding 5 ml of a solution of 0.02 M 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS) in 15% acetic acid and 5 ml of 0.1 M sodium cyanoborohydride in DMSO to the dried samples. The ANTS-labeled products were then dried and resuspended in 25 ml of loading dye (0.015% of bromophenol blue plus 10% glycerol in 62 mM Tris-HCl pH 6.8). Approximately 0.5-1 mg of ANTS-labeled product were loaded onto a 35% polyacrylamide (19:1) gel with a 10% stacking gel and electrophoresed at a constant 100 V for 30 min followed by 1 hr at 300 V at 4 C in native running buffer (25 mM Tris-HCl, 0.2 M glycine). Gels were immediately visualized and imaged under UV light. A time-course digestion of laminarin was also analyzed by FACE. 50 nM of enzyme was incubated in presence of 1% laminarin in 20 mM Tris-HCl pH 8.0 over 40 min. At specific time points, a 15 ml aliquot was taken and the reaction stopped into 1 ml of ice cold 100% ethanol. The samples were then dried, labeled and visualized as described above.

e2 Structure 25, 1–12.e1–e3, September 5, 2017

Please cite this article in press as: Pluvinage et al., Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of b-1,3-Glucan Quaternary Structure, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.019

Enzyme Kinetic Assays Steady state kinetics studies were performed using the 3,5-dinitrosalicylic acid assay adapted from McKee (McKee, 2017) and read on a SpectraMax M5 spectrophotometer. Standard reaction mixtures were done in 20 mM Tris-HCl buffer, pH 8.0 containing 200 nM enzyme and 0 – 18 mg/ml laminarin or 2 mM enzyme and 0 – 24 mg/ml curdlan. 3,5-dinitroslicylic acid (DNSA) reagent (1% DNSA, 0.2% (v/v) phenol, 0.05% (w/v) Na2SO3 and 1% NaOH) supplemented with 0.02% glucose, was used to quantify the reducing sugars by reading the absorbance at 575 nm. Glucose ranging from 0 to 1.6 mg/ml was used to generate a standard curve; using this curve the absorbance reading of samples were converted to the concentration of glucose equivalents. General Crystallography Procedures Crystals were obtained using sitting-drop vapor diffusion for screening and hanging drop vapor diffusion for optimization, all at 18 C. For data collection single crystals were flash cooled with liquid nitrogen in crystallization solution supplemented with a cryoprotectant optimized for each crystal form as given below. Diffraction data were collected either on an ‘‘in-house’’ beam comprising a Pilatus 200K 2D detector coupled to a MicroMax-007HF X-ray generator with a VariMaxTM-HF Arc)Sec Confocal Optical System and an Oxford Cryostream 800, beamline 11-1 at Stanford Linear Accelerator Center (SLAC, Stanford Synchrotron Radiation Lightsource [SSRL], California), or beamline 08ID-1 at the Canadian Light Source (CLS, Saskatoon, Saskatchewan) as indicated in Tables S1 and S2. All diffraction data were processed using MOSFLM and SCALA (Collaborative Computational Project, Number 4, 1994; Powell, 1999). All data collection and processing statistics are shown in Tables S1 and S2. For all structures, manual model building was performed with COOT (Emsley and Cowtan, 2004) and refinement of atomic coordinates was performed with REFMAC (Murshudov et al., 1997). The addition of water molecules was performed in COOT with FINDWATERS and manually checked after refinement. In all data sets, refinement procedures were monitored by flagging 5% of all observation as ‘‘free’’ (Brunger, 1992). Model validation was performed with MOLPROBITY (Davis et al., 2007). All model statistics are shown in Tables S1 and S2. Coordinates and structure factors have been deposited with the PDB codes indicated below and in the Key resources Table. BhGH81 Structure Determination Crystals of BhGH81 seleno-methionine derivative (72 mg/ml) were obtained in 1.4 / 0.35 M sodium phosphate monobasic / potassium phosphate dibasic (NaH2PO4 / K2HPO4) and 0.075 M sodium phosphate dibasic (Na2HPO4) / citric acid, pH 3.2. Crystals were cryoprotected using 25% (v/v) ethylene glycol prior to freezing. The heavy atom substructure comprising 16 seleno-methionine residues, refinement, phasing, and density modification was performed using autoSHARP (Vonrhein et al., 2007). An initial model was then built using ARP/wARP (Langer et al., 2008). Crystals of BhGH81 (30 mg/ml) were grown in 20% polyethylene glycol (PEG) 3,350 and 0.2 M sodium iodide. These crystals were soaked in crystallization solution supplemented with an excess of L6 for 20 min. Crystals of BhGH81 (50 - 70 mg/ml) were also obtained in 1.2 - 1.4 M NaH2PO4, 0.2 M K2HPO4 and 0.1 M citric acid pH 4.2. These crystals were soaked in crystallization solution containing an excess of L2 for 20 min or containing 10% laminarin for a month. After soaking, crystals were frozen using 25% (v/v) ethylene glycol in the crystallization solution as cryoprotectant. Crystals of BhGH81E542Q (70 mg/ml) were grown in 15% PEG 10,000, 0.2 M ammonium acetate and 0.1 M Bis-Tris/HCl, pH 5.5. These crystals were soaked for 30 min in crystallization solution supplemented with an excess of L2, L3, L4, L5, or L6 prior to freezing using 25% (v/v) ethylene glycol in crystallization solution. The model built with the BhGH81 seleno-methionine derivative data was used to solve the structures of BhGH81 and BhGH81E542Q by molecular replacement using PHASER (McCoy et al., 2007). DATA AND SOFTWARE AVAILABILITY BhGH81 seleno-methionine derivative coordinates have been deposited with the PDB ID 5T49. The coordinates of BhGH81 in complex with laminarin, laminarihexaose and laminaribiose have been deposited with the PDB ID 5T4G, 5T4A, and 5V1W, respectively. The coordinates of GH81E542Q in complex with laminaribiose, laminaritriose, laminaritetraose, laminaripentaose and laminarihexaose have been deposited with the PDB ID 5UPI, 5UPM, 5UPN, 5UPO and 5T4C, respectively (See Key Resources Table).

Structure 25, 1–12.e1–e3, September 5, 2017 e3