Distinct protein architectures mediate species-specific beta-glucan binding and metabolism in the human gut microbiota

Complex glycans that evade our digestive system are major nutrients that feed the human gut microbiota (HGM). The prevalence of Bacteroidetes in the HGM of populations worldwide is engendered by the evolution of polysaccharide utilization loci (PULs), which encode concerted protein systems to utilize the myriad complex glycans in our diets. Despite their crucial roles in glycan recognition and transport, cell-surface glycan-binding proteins (SGBPs) remained understudied cogs in the PUL machinery. Here, we report the structural and biochemical characterization of a suite of SGBP-A and SGBP-B structures from three syntenic β(1,3)-glucan utilization loci (1,3GULs) from Bacteroides thetaiotaomicron (Bt), Bacteroides uniformis (Bu), and B. fluxus (Bf), which have varying specificities for distinct β-glucans. Ligand complexes provide definitive insight into β(1,3)-glucan selectivity in the HGM, including structural features enabling dual β(1,3)-glucan/mixed-linkage β(1,3)/β(1,4)-glucan-binding capability in some orthologs. The tertiary structural conservation of SusD-like SGBPs-A is juxtaposed with the diverse architectures and binding modes of the SGBPs-B. Specifically, the structures of the trimodular BtSGBP-B and BuSGBP-B revealed a tandem repeat of carbohydrate-binding module-like domains connected by long linkers. In contrast, BfSGBP-B comprises a bimodular architecture with a distinct β-barrel domain at the C terminus that bears a shallow binding canyon. The molecular insights obtained here contribute to our fundamental understanding of HGM function, which in turn may inform tailored microbial intervention therapies. Complex glycans that evade our digestive system are major nutrients that feed the human gut microbiota (HGM). The prevalence of Bacteroidetes in the HGM of populations worldwide is engendered by the evolution of polysaccharide utilization loci (PULs), which encode concerted protein systems to utilize the myriad complex glycans in our diets. Despite their crucial roles in glycan recognition and transport, cell-surface glycan-binding proteins (SGBPs) remained understudied cogs in the PUL machinery. Here, we report the structural and biochemical characterization of a suite of SGBP-A and SGBP-B structures from three syntenic β(1,3)-glucan utilization loci (1,3GULs) from Bacteroides thetaiotaomicron (Bt), Bacteroides uniformis (Bu), and B. fluxus (Bf), which have varying specificities for distinct β-glucans. Ligand complexes provide definitive insight into β(1,3)-glucan selectivity in the HGM, including structural features enabling dual β(1,3)-glucan/mixed-linkage β(1,3)/β(1,4)-glucan-binding capability in some orthologs. The tertiary structural conservation of SusD-like SGBPs-A is juxtaposed with the diverse architectures and binding modes of the SGBPs-B. Specifically, the structures of the trimodular BtSGBP-B and BuSGBP-B revealed a tandem repeat of carbohydrate-binding module-like domains connected by long linkers. In contrast, BfSGBP-B comprises a bimodular architecture with a distinct β-barrel domain at the C terminus that bears a shallow binding canyon. The molecular insights obtained here contribute to our fundamental understanding of HGM function, which in turn may inform tailored microbial intervention therapies. The distal human gastrointestinal tract plays host to a highly dynamic community of microbes, collectively known as the human gut microbiota (HGM). Strong correlational and causal links between composition of the HGM and numerous disease states have been established (1Ridaura V.K. Faith J.J. Rey F.E. Cheng J.Y. Duncan A.E. Kau A.L. Griffin N.W. Lombard V. Henrissat B. Bain J.R. 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Surface glycan-binding proteins are essential for cereal beta-glucan utilization by the human gut symbiont Bacteroides ovatus.Cell. Mol. Life Sci. 2019; 76: 4319-4340Crossref PubMed Scopus (20) Google Scholar), β(1,3)-glucan (from fungi/yeasts, plants) (25Déjean G. Tamura K. Cabrera A. Jain N. Pudlo N.A. Pereira G. Viborg A.H. Van Petegem F. Martens E.C. Brumer H. Synergy between cell surface glycosidases and glycan-binding proteins dictates the utilization of specific beta(1,3)-glucans by human gut.mBio. 2020; 11e00095-20Crossref PubMed Scopus (27) Google Scholar), and β(1,6)-glucan (from fungi) (36Temple M.J. Cuskin F. Basle A. Hickey N. Speciale G. Williams S.J. Gilbert H.J. Lowe E.C. A Bacteroidetes locus dedicated to fungal 1,6--glucan degradation: Unique substrate conformation drives specificity of the key endo-1,6--glucanase.J. Biol. Chem. 2017; 292: 10639-10650Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In our recent study of homologous β(1,3)-glucan utilization loci (1,3GULs) from Bacteroides uniformis, B. thetaiotaomicron, and B. fluxus, we demonstrated that growth on individual branched β(1,3)-glucans and/or MLG is dependent on the combinatorial specificities of cognate GHs and SGBPs encoded by each species. (Fig. 1) (25Déjean G. Tamura K. Cabrera A. Jain N. Pudlo N.A. Pereira G. Viborg A.H. Van Petegem F. Martens E.C. Brumer H. Synergy between cell surface glycosidases and glycan-binding proteins dictates the utilization of specific beta(1,3)-glucans by human gut.mBio. 2020; 11e00095-20Crossref PubMed Scopus (27) Google Scholar). Here, we explore the structural basis underlying the specificity of the three orthologous SGBPs-A (SusD homologs) and the three sequence-diverse SGBPs-B through X-ray crystallography and isothermal titration calorimetry with defined oligosaccharides. In doing so, we uncover unique structural architectures that allow recognition of distinct classes of β-glucan. PULs classically encode two distinct types of SGBPs. SGBPs-A (also referred to as SusD-like or SusD-homologs) are highly conserved across PUL systems, in which they form the extracellular lid of an active transport complex with the cognate TBDT (SusC homologs) (27Bolam D.N. Koropatkin N.M. Glycan recognition by the Bacteroidetes sus-like systems.Curr. Opin. Struct. Biol. 2012; 22: 563-569Crossref PubMed Scopus (43) Google Scholar, 28Glenwright A.J. Pothula K.R. Bhamidimarri S.P. Chorev D.S. Basle A. Firbank S.J. Zheng H.J. Robinson C.V. Winterhalter M. Kleinekathofer U. Bolam D.N. van den Berg B. Structural basis for nutrient acquisition by dominant members of the human gut microbiota.Nature. 2017; 541: 407-411Crossref PubMed Scopus (108) Google Scholar). Indeed, the tandem TBDT/SGBP-A (susC/susD homolog) gene pair is a signature feature used to identify PULs in sequenced Bacteroidetes genomes (12Martens E.C. Lowe E.C. Chiang H. Pudlo N.A. Wu M. McNulty N.P. Abbott D.W. Henrissat B. Gilbert H.J. Bolam D.N. Gordon J.I. Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts.PLoS Biol. 2011; 9: 1-16Crossref Scopus (464) Google Scholar, 37Terrapon N. Lombard V. Drula E. Lapebie P. Al-Masaudi S. Gilbert H.J. Henrissat B. Puldb: The expanded database of polysaccharide utilization loci.Nucleic Acids Res. 2018; 46: D677-D683Crossref PubMed Scopus (91) Google Scholar). On the other hand, PULs often encode one or more additional, often highly sequence-divergent, SGBP(s) immediately downstream of the SGBP-A (susD) homolog, here denoted as SGBP-B (30Tauzin A.S. Kwiatkowski K.J. Orlovsky N.I. Smith C.J. Creagh A.L. Haynes C.A. Wawrzak Z. Brumer H. Koropatkin N.M. Molecular dissection of xyloglucan recognition in a prominent human gut symbiont.mBio. 2016; 7: 15Crossref Scopus (49) Google Scholar, 32Tamura K. Foley M.H. Gardill B.R. Dejean G. Schnizlein M. Bahr C.M.E. Louise Creagh A. van Petegem F. Koropatkin N.M. Brumer H. Surface glycan-binding proteins are essential for cereal beta-glucan utilization by the human gut symbiont Bacteroides ovatus.Cell. Mol. Life Sci. 2019; 76: 4319-4340Crossref PubMed Scopus (20) Google Scholar). These are sometimes referred to a "SusE-positioned" proteins due to this genetic organization but lack of sequence similarity with SusE (29Cameron E.A. Maynard M.A. Smith C.J. Smith T.J. Koropatkin N.M. Martens E.C. Multidomain carbohydrate-binding proteins involved in Bacteroides thetaiotaomicron starch metabolism.J. Biol. Chem. 2012; 287: 34614-34625Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The 1,3GULs from B. uniformis ATCC8492, B. thetaiotaomicron NLAE-zl-H207, and B. fluxus YIT12057 each encode one SGBP-A and one SGBP-B (Fig. 1, B and C), the tertiary structures of which we determined in free and oligosaccharide-complexed forms. All three SGBPs-A are predicted to be outer membrane surface-anchored via a cysteine lipidation at the N terminus of their respective mature polypeptides (Cys22 in BuSGBP-A, Cys21 in BtSGBP-A, Cys22 in BfSGBP-A (38Juncker A.S. Willenbrock H. Von Heijne G. Brunak S. Nielsen H. Krogh A. Prediction of lipoprotein signal peptides in Gram-negative bacteria.Protein Sci. 2003; 12: 1652-1662Crossref PubMed Scopus (850) Google Scholar, 39Nielsen H. Predicting secretory proteins with SignalP.Methods Mol. Biol. 2017; 1611: 59-73Crossref PubMed Scopus (499) Google Scholar)). Hence, the recombinant forms of these proteins were produced with both the native signal peptide and the cysteine removed. Diffraction quality crystals of BuSGBP-A, BtSGBP-A, and BfSGBP-A were successfully grown following varying amounts of optimization. Crystals of BtSGBP-A required micro seeding to reproduce crystallization screen hits, while the morphology and diffraction quality of BuSGBP-A were significantly improved by the addition of hexamminecobalt(III) chloride (see Experimental procedures and Fig. S1A). Experimental phases for BuSGBP-A were obtained by single anomalous dispersion at the cobalt absorption edge (Table S1). There are two ordered cobalt sites with significant anomalous signal, one per each of two molecules in the asymmetric unit. Both hexamminecobalt(III) complexes were found to mediate crystal contacts to molecules in neighboring asymmetric units, explaining the improved diffraction (Fig. S1, B and C). Coordinates were refined to a final resolution of 1.85 Å against a higher resolution data set collected at shorter wavelength (Table S1). The completed model of both molecules in the asymmetric unit comprised residues 43 to 529 with unmodeled gaps at residues 176 to 177, 211 to 219, 293 to 308, and 390 to 394, due to lack of corresponding electron density (Fig. 2A). The crystal structures of BtSGBP-A and BfSGBP-A were solved to 1.80 Å and 1.84 Å, respectively, by molecular replacement using the BuSGBP-A structure as a search model (Table S1). Both consist of a single molecule in their respective asymmetric units and the resulting electron density allowed near-complete tracing of the protein model comprising residues 38 to 515 for BtSGBP-A and residues 40 to 510 for BfSGBP-A (Fig. 2A). As observed for BuSGBP-A, the first ca. 20 amino acids in both proteins were disordered, suggesting that these form flexible tethers from the cell surface in the native lipoproteins. Otherwise, the only unmodeled gaps in the BtSGBP-A and BfSGBP-A structures correspond to residues 172 to 179 and residues 172 to 175, respectively. The C termini were defined in the electron density of all three SGBPs-A. The three orthologous SGBPs-A were all observed to possess the canonical SusD fold with tetratricopeptide repeats (TPRs) prominently forming the structural scaffold (Fig. 2A) (40Koropatkin N.M. Martens E.C. Gordon J.I. Smith T.J. Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices.Structure. 2008; 16: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The structures are almost identical and superpose with low pairwise root-mean-square deviation (RMSD) values, which negatively correlate with amino acid sequence identity, as expected (Fig. 2B, Table S2). One prominent difference is the insertion of an α-helix at the periphery of BtSGBP-A (residues 346–353), which is not present in BuSGBP-A nor BfSGBP-A. The functional significance of this additional helix, if any, is unknown. Other differences in the observed residues are restricted to minor variations in the positioning of surface loops. Soaking of the native BtSGBP-A with laminarihexaose yielded a 2.05 Å ligand-complexed structure (Table S1) that clearly revealed an extended β(1,3)-glucan-binding platform, notably comprising two key tryptophan residues (Trp288 and Trp318) and specific recognition of the nonreducing end by multiple hydrogen bonds (Fig. 2). Electron density was observed for all six glucosyl residues, which were convincingly modeled in the favored 4C1 conformation (Fig. 2C, Table S2). The bound laminarihexaose molecule adopts an extended helical structure with the binding surface complementing a low-energy conformation of the oligosaccharide (41Bacic A. Fincher G.B. Stone B.A. Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides. Academic Press, Cambridge, MA2009Google Scholar). As is typical for β(1,3)-glucans, hydrogen bonds between the ring oxygen and the C4-OH of the adjacent glucosyl residue are observed, except between Glc3 and Glc4. Here, there is a ca. 180° flip in the ϕ (O5-C1-O3'-C3') angle of the glycosidic bond between Glc3 and Glc4, imparting an additional twist in the helix. BtSGBP-A residues involved in binding interactions with the hexasaccharide are borne on a polypeptide connecting the two α-helices comprising TPR1 (residues 59–107) and the TPR3 and TPR4 domains (residues 279–398). A bis-tris molecule observed in the original native structure was displaced by the nonreducing-end glucose (Fig. S2), which displays a very favorable carbohydrate–aromatic interaction with Trp319 (Fig. 2, D–F). In addition, four hydrogen bonds between C3-OH and Arg367-Nη1, C4-OH and Arg367-Nη2, C4-OH and Asp90-Oδ1, and C6-OH and Asp90-Oδ2 firmly anchor the nonreducing-end glucose in place. The strength of these collective interactions is reflected in the comparatively very low B-factor of Glc6 (Fig. 2D). In this context it is also worth noting that Tyr306 is well positioned to firmly hold Arg367 in place via a π–cation interaction. Trp288 partially stacks against both Glc5 and Glc4, with the former also hydrogen bonding to Tyr67 via the C4-OH, and the latter exhibiting water-mediated hydrogen bonding to Glu393 via the C2-OH (Fig. 2E). The only interaction to Glc3 is a hydrogen bond between Ser391 and the C2-OH resulting in a higher B-factor on the C5 side of the ring (Fig. 2D). Glc2 and Glc1 both hydrogen-bond to the same Oδ of Asp287 via their respective C2-OH groups (Fig. 2E). Additional hydrogen bonds were observed between Ser390 and the glycosidic oxygen connecting Glc2 and Glc1, as well as between the main chain carbonyl oxygen of Lys389 and the C4-OH of Glc1. Finally, a possible CH–π interaction between Glc2 and the amide bond between Ser390 and Ser391, as well as hydrophobic interaction between Glc1 and Val285, may also contribute to binding at these sites (Fig. 2E). Overall, the native and laminarihexaose-complexed structures of BtSGBP-A are virtually identical, superposing with an RMSD value of 0.19 Å over 402 Cα pairs (Fig. S2C). The key binding site residues are also in essentially identical positions except for Ser391 and Glu393, which upon binding laminarihexaose rotate to participate in direct and water-mediated hydrogen bonding, respectively (Fig. 2E). Despite BtSGBP-A binding the nonreducing end of the glycan chain in a manner reminiscent of a type-C carbohydrate-binding module (CBM) (42Gilbert H.J. Knox J.P. Boraston A.B. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules.Curr. Opin. Struct. Biol. 2013; 23: 669-677Crossref PubMed Scopus (208) Google Scholar), the binding pocket is not prominent and rather constitutes a binding platform that is blocked on one end (Fig. 2, C and D). Indeed, this extended binding site requires a β(1,3)-glucan substrate with a degree of polymerization (DP) ≥ 5 to effect binding, as revealed by isothermal titration calorimetry (ITC). BtSGBP-A bound laminarihexaose (KD 26 μM) approximately tenfold tighter than laminaripentaose (KD 210 μM), whereas binding to laminaritriose and laminaritetraose was too weak to be quantified (Fig. S4, Table S4). Likewise, soaking native BtSGBP-A crystals with laminaritriose did not yield a trisaccharide-complexed structure. In the biological context, our previous affinity gel electrophoresis (AGE) and ITC analyses using polysaccharides demonstrated that BtSGBP-A is responsible for binding β(1,3)-glucans with varying degrees and lengths of β(1,6)-linked glucosyl branching (25Déjean G. Tamura K. Cabrera A. Jain N. Pudlo N.A. Pereira G. Viborg A.H. Van Petegem F. Martens E.C. Brumer H. Synergy between cell surface glycosidases and glycan-binding proteins dictates the utilization of specific beta(1,3)-glucans by human gut.mBio. 2020; 11e00095-20Crossref PubMed Scopus (27) Google Scholar). The laminarihexaose complex here reveals the abundance of space around the C6-OH group at every glucose binding subsite, thereby rationalizing this versatility (Fig. 2D). Despite extensive efforts, including soaking and cocrystallization trials, we were not able to obtain a structure of BfSGBP-A in complex with a laminarioligosaccharide. However, sequence alignment combined with structural superposition reveals that many of the key binding site res