Unique properties of a Dictyostelium discoideum carbohydrate-binding module expand our understanding of CBM–ligand interactions

Abstract

Deciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine. Here, we present the first crystallographic structure of a CBM8 family member (CBM8), DdCBM8, from the slime mold Dictyostelium discoideum, which was identified attached to an endo-β-1,4-glucanase (glycoside hydrolase family 9). We show that the planar carbohydrate-binding site of DdCBM8, composed of aromatic residues, is similar to type A CBMs that are specific for crystalline (multichain) polysaccharides. Accordingly, pull-down assays indicated that DdCBM8 was able to bind insoluble forms of cellulose. However, affinity gel electrophoresis demonstrated that DdCBM8 also bound to soluble (single chain) polysaccharides, especially glucomannan, similar to type B CBMs, although it had no apparent affinity for oligosaccharides. Therefore, the structural characteristics and broad specificity of DdCBM8 represent exceptions to the canonical CBM classification. In addition, mutational analysis identified specific amino acid residues involved in ligand recognition, which are conserved throughout the CBM8 family. This advancement in the structural and functional characterization of CBMs contributes to our understanding of carbohydrate-active enzymes and protein–carbohydrate interactions, pushing forward protein engineering strategies and enhancing the potential biotechnological applications of glycoside hydrolase accessory modules. Deciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine. Here, we present the first crystallographic structure of a CBM8 family member (CBM8), DdCBM8, from the slime mold Dictyostelium discoideum, which was identified attached to an endo-β-1,4-glucanase (glycoside hydrolase family 9). We show that the planar carbohydrate-binding site of DdCBM8, composed of aromatic residues, is similar to type A CBMs that are specific for crystalline (multichain) polysaccharides. Accordingly, pull-down assays indicated that DdCBM8 was able to bind insoluble forms of cellulose. However, affinity gel electrophoresis demonstrated that DdCBM8 also bound to soluble (single chain) polysaccharides, especially glucomannan, similar to type B CBMs, although it had no apparent affinity for oligosaccharides. Therefore, the structural characteristics and broad specificity of DdCBM8 represent exceptions to the canonical CBM classification. In addition, mutational analysis identified specific amino acid residues involved in ligand recognition, which are conserved throughout the CBM8 family. This advancement in the structural and functional characterization of CBMs contributes to our understanding of carbohydrate-active enzymes and protein–carbohydrate interactions, pushing forward protein engineering strategies and enhancing the potential biotechnological applications of glycoside hydrolase accessory modules. Glycoside hydrolases (GHs) are enzymes capable of breaking glycosidic bonds. They are found in all living beings and are involved in essential functions, such as cell wall modeling, defense, symbiosis, signaling, biosynthesis, and nutrient acquisition (1Minic Z. Physiological roles of plant glycoside hydrolases.Planta. 2008; 227: 723-740Crossref PubMed Scopus (199) Google Scholar). These enzymes are broadly applied in the industrial production of paper, fabrics, and food and have gained visibility in biorefining processes for conversion of biomass into renewable fuels and chemicals (2Kirk O. Borchert T.V. Fuglsang C.C. Industrial enzyme applications.Curr. Opin. Biotechnol. 2002; 13: 345-351Crossref PubMed Scopus (988) Google Scholar, 3Linares-Pasten J. Andersson M. Karlsson E. Thermostable glycoside hydrolases in biorefinery technologies.Curr. Biotechnol. 2014; 3: 26-44Crossref Google Scholar, 4Consortium T. Caz Ten years of CAZypedia: A living encyclopedia of carbohydrate-active enzymes.Glycobiology. 2018; 28: 3-8Crossref PubMed Scopus (93) Google Scholar). GHs are composed of a catalytic domain, which is often covalently linked to one or more accessory modules that regulate their activity, such as carbohydrate-binding modules (CBMs). CBMs are discrete folding units capable of binding to different types of carbohydrates, and their main role is to mediate the interaction between the enzyme and the target substrate, leading to modifications in catalytic efficiency (5Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Carbohydrate-binding modules: Fine-tuning polysaccharide recognition.Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1459) Google Scholar). More specifically, CBMs perform functions such as increasing substrate accessibility through disruption of the crystalline structure of cellulose (6Din N. Gilkes N.R. Tekant B. Miller R.C. Warren R.A.J. Kilburn D.G. 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Rep. 2016; 6: 23473Crossref PubMed Scopus (18) Google Scholar). To date, CBMs have been grouped into 89 different families in the carbohydrate-active enzymes (CAZy) database based on amino acid sequence similarities (10Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42: D490-D495Crossref PubMed Scopus (3787) Google Scholar). CBMs are also classified based on their functional properties: type A CBMs possess flat binding faces, capable of binding to crystalline polysaccharides; type B CBMs bind internally to soluble polysaccharides via cleft-shaped contact sites; and type C CBMs interact with the terminal regions of carbohydrates through protein pocket–shaped sites (5Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Carbohydrate-binding modules: Fine-tuning polysaccharide recognition.Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1459) Google Scholar, 11Gilbert H.J. Paul Knox J. 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 (209) Google Scholar). CBMs are considered auspicious for various biotechnological purposes, such as modification of the physical properties of fibers and potentiating enzymatic degradation of polysaccharides (12Mello B.L. Polikarpov I. Family 1 carbohydrate binding-modules enhance saccharification rates.AMB Express. 2014; 4: 30Crossref PubMed Scopus (21) Google Scholar, 13Nagl M. Haske-Cornelius O. Skopek L. Bausch F. Pellis A. Bauer W. Nyanhongo G.S. Guebitz G.M. Mechanistic investigation of the effect of endoglucanases related to pulp refining.Cellulose. 2022; https://doi.org/10.1007/S10570-021-04386-5Crossref Google Scholar), suitable for application in textile, food, and feed industries. CBMs are valuable tools in basic science research, used on in situ visualizations of polysaccharides, in vivo expression in plant physiology studies (14Mei X. Chang Y. Shen J. Zhang Y. Xue C. Expression and characterization of a novel alginate-binding protein: A promising tool for investigating alginate.Carbohydr. Polym. 2020; 246: 116645Crossref PubMed Scopus (3) Google Scholar), and high-throughput analysis of polysaccharides based on microarrays (15Moller I. Sørensen I. Bernal A.J. Blaukopf C. Lee K. Øbro J. Pettolino F. Roberts A. Mikkelsen J.D. Knox J.P. Bacic A. Willats W.G.T. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays.Plant J. 2007; 50: 1118-1128Crossref PubMed Scopus (242) Google Scholar). In synthetic biology initiatives, these protein modules are used as building blocks to construct minicellulosomes (16Kim S. Baek S.H. Lee K. Hahn J.S. Cellulosic ethanol production using a yeast consortium displaying a minicellulosome and β-glucosidase.Microb. Cell Fact. 2013; 12: 14Crossref PubMed Scopus (64) Google Scholar). In biomedicine, CBMs are employed to functionalize carbohydrate-based biomaterial and improve recombinant protein technology, promoting expression, purification, stabilization, and immobilization of heterologous proteins (17Oliveira C. Carvalho V. Domingues L. Gama F.M. Recombinant CBM-fusion technology - applications overview.Biotechnol. Adv. 2015; 33: 358-369Crossref PubMed Scopus (85) Google Scholar, 18Qin Z. Lin S. Qiu Y. Chen Q. Zhang Y. Zhou J. Zhao L. One-step immobilization-purification of enzymes by carbohydrate-binding module family 56 tag fusion.Food Chem. 2019; 299: 125037Crossref PubMed Scopus (21) Google Scholar, 19Shoseyov O. Shani Z. Levy I. Carbohydrate binding modules: Biochemical properties and novel applications.Microbiol. Mol. Biol. Rev. 2006; 70: 283-295Crossref PubMed Scopus (408) Google Scholar). In this sense, the CBMs were employed for expression at the surface of proteins of pathogens (17Oliveira C. Carvalho V. Domingues L. Gama F.M. Recombinant CBM-fusion technology - applications overview.Biotechnol. Adv. 2015; 33: 358-369Crossref PubMed Scopus (85) Google Scholar), such as antigenic protein fragments of severe acute respiratory syndrome coronavirus 2 (20McGuire B.E. Mela J.E. Thompson V.C. Cucksey L.R. Stevens C.E. McWhinnie R.L. Winkler D.F.H. Pelech S. Nano F.E. Escherichia coli recombinant expression of SARS-CoV-2 protein fragments.Microb. Cell Fact. 2022; 21: 21Crossref PubMed Scopus (0) Google Scholar). The number of different sequences deposited in CAZy and the number of families have been rapidly increasing because of next-generation sequencing technologies (21Kameshwar A.K.S. Qin W. Recent developments in using advanced sequencing technologies for the genomic studies of lignin and cellulose degrading microorganisms.Int. J. Biol. Sci. 2016; 12: 156-171Crossref PubMed Scopus (57) Google Scholar). Occasionally, members from novel or poorly studied families reveal new characteristics that show uncertainties in the current classifications (22Campos B.M. Liberato M.V. Alvarez T.M. Zanphorlin L.M. Ematsu G.C. Barud H. Polikarpov I. Ruller R. Gilbert H.J. De Mattos Zeri A.C. Squina F.M. A novel carbohydrate-binding module from sugar cane soil metagenome featuring unique structural and carbohydrate affinity properties ∗.J. Biol. Chem. 2016; 291: 23734-23743Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) as well as disclose opportunities to be explored for biotechnological purposes. Therefore, we selected CBM8 (DdCBM8) from the endo-β-1,4-glucanase (CelA) of Dictyostelium discoideum (Fig. 1). DdCBM8 is located at the C-terminal region of CelA and is connected to the catalytic GH9 domain through a threonine–glutamate–threonine–proline repeat linker (23Ramalingam R. Blume J.E. Ennis The Dictyostelium discoideum is into functional PubMed Google Scholar). to our the DdCBM8 a planar binding site and has the of binding to insoluble crystalline type A CBM However, DdCBM8 affinity for soluble polysaccharides, such as glucomannan, type B We to the DdCBM8 using and to the unique structural and functional properties to novel into that be for protein engineering increasing the biotechnological application of accessory modules. The DdCBM8, amino acid residues to from CelA (Fig. was into the bacterial in soluble using Escherichia coli as the and to using affinity and a pull-down was to DdCBM8 to insoluble polysaccharides. of DdCBM8 were with of and bacterial cellulose the protein bound to the polysaccharides was with in DdCBM8 was able to bind to the polysaccharides is present in the insoluble to the affinity to insoluble polysaccharides was using different of DdCBM8 were with of and the of bound protein reveal the affinity In the binding was (Fig. However, was of the affinity of DdCBM8 for and of binding of DdCBM8 to soluble and insoluble polysaccharides. of DdCBM8 binding to insoluble forms of cellulose and of in the and of different soluble polysaccharide and The the of by of increasing of of from the of DdCBM8 by soluble polysaccharides glucomannan, or carbohydrate-binding bacterial The of DdCBM8 to bind soluble polysaccharides was using an affinity gel electrophoresis with different polysaccharides were used in and the protein was binding between protein and in to a gel the ligand in DdCBM8 bound to glucomannan, and cellulose to was to (Fig. and the binding of DdCBM8 to these polysaccharides (Fig. and The were to a binding The molecular of polysaccharides was employed to the binding to these polysaccharides, although this the affinity as often bind The DdCBM8 binding for was for and was and for was also as no was by to the binding affinity was no binding of DdCBM8 to or such as and or for a with and an be based on and DdCBM8 crystallographic structure was at were using the using of as in the the of the of with from to The for and are in The structure a in the and all amino acid residues were with The amino terminal also residues and from the DdCBM8 has a globular of the CBMs, and composed of with and and and and that are connected by and one (Fig. A structural with L. mapping in Acids Res. PubMed Scopus Google Scholar) and E. K. a new tool for protein structure in Biol. 2004; PubMed Scopus Google Scholar) that DdCBM8 with members of CBM with with and with to and with and and which is with the of affinity for these However, aromatic residues to and a planar surface a site in type A CBMs (Fig. the structural with CBMs, these aromatic residues were with the binding site of a member of the Bolam D.N. L. Gilbert H.J. Davies G.J. in The structure of a carbohydrate-binding in with and Sci. S. A. 2002; PubMed Scopus Google Scholar), which was with CBMs and present the aromatic residues in similar which in the of were for ligand binding through residues that are involved in ligand through in and from DdCBM8 and and from (Fig. The main in the binding sites is the of amino and in which interact with the ligand through in DdCBM8 has residues with that in which is for a the ligand (Fig. The found in DdCBM8 a planar for this region that crystalline polysaccharide To the site from the crystallographic structure and to the role of amino acid in the residues of the sites were to and and the CBMs were to assays using for DdCBM8 (Fig. from the crystallographic in amino that the of the binding site through and DdCBM8 from binding to all polysaccharides The be for the of we were able to the specific protein was from that and interact with the to be in a for with the as indicated by the in the effect was to an in ligand role on the binding The amino acid sequence of DdCBM8 was with members of the CBM8 family (Fig. In the sequences However, the residues that the binding site and were The residues and which to our protein–carbohydrate were conserved CBM8 family the amino acid residues involved in binding and the conserved found in the are in the protein structure and to role in CBM A (Fig. with DdCBM8 and the members in family Bolam D.N. L. Gilbert H.J. Davies G.J. in The structure of a carbohydrate-binding in with and Sci. S. A. 2002; PubMed Scopus Google Scholar, J. Bolam D.N. Williamson M.P. C. Davies G.J. Gilbert H.J. the of ligand recognition in family carbohydrate-binding Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the aromatic residues the binding site were conserved as However, the CBM8 and family members sequence it is to The CBM8 family has members to date, of which to and the to discoideum, which is a slime mold considered as a and to signaling, and interaction similarities with J. J. Dictyostelium discoideum as a Biotechnol. PubMed Scopus Google Scholar, J. J. Dictyostelium discoideum as a to the of and J. Biol. 2019; PubMed Scopus Google Scholar). The first and unique of this family is related to the of an endo-β-1,4-glucanase from discoideum, CelA (23Ramalingam R. Blume J.E. Ennis The Dictyostelium discoideum is into functional PubMed Google Scholar). CelA was able to bind to the domain or the with linker region M. Haske-Cornelius O. Skopek L. Bausch F. Pellis A. Bauer W. Nyanhongo G.S. Guebitz G.M. Mechanistic investigation of the effect of endoglucanases related to pulp refining.Cellulose. 2022; https://doi.org/10.1007/S10570-021-04386-5Crossref Google Scholar). the C-terminal domain from CelA in this as was for the (23Ramalingam R. Blume J.E. Ennis The Dictyostelium discoideum is into functional PubMed Google Scholar), the pull-down the affinity of DdCBM8 for and that it was able to bind to as has a of crystalline cellulose as with M. Lee - a of the enzymatic J. PubMed Scopus Google Scholar), we that DdCBM8 has affinity for the crystalline However, the that this affinity is that for type A CBMs of a novel family of carbohydrate-binding modules with broad ligand Rep. 2016; 6: PubMed Scopus Google Scholar, N. basis for cellulose binding by a module and bacterial Sci. S. A. PubMed Scopus Google Scholar, M. V. S. I. Carvalho and ligand of type A carbohydrate-binding modules are by the structure of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it was that DdCBM8 had a affinity for soluble polysaccharides, especially The binding for is similar to that found for type B CBMs to Bolam D.N. L. Gilbert H.J. Davies G.J. in The structure of a carbohydrate-binding in with and Sci. S. A. 2002; PubMed Scopus Google I. M. J. S. A. Coutinho P.M. Henrissat B. Knox J.P. A. of the an in Sci. S. A. 2016; PubMed Scopus Google Scholar). the to an in DdCBM8 as a type A or B. The present is the first structure of a CBM8 family to The crystallographic structure of DdCBM8 folding of the CBMs, no similar structure was found in binding site was by structural with members of the family and via of amino The planar binding site in the DdCBM8 structure to as a type A However, similar to DdCBM8 is to canonical classification. be a type B with affinity for soluble (single chain) polysaccharides or type A to the planar binding site of the However, DdCBM8 binding to and has a affinity for crystalline cellulose. The unique characteristics of DdCBM8, with broad to a understanding of CBM and biotechnological The structure of DdCBM8 has no in CBM which has a planar binding site that to as a type A However, DdCBM8 binding to and has a affinity for crystalline which are functional properties of type A the DdCBM8 type B functional characteristics because of the affinity for soluble (single chain) polysaccharides. Therefore, the structural characteristics and broad specificity of DdCBM8 and this canonical and on the current understanding of CBM interaction and classification. The the CBM DdCBM8, found in an from discoideum was by from to The the was with and enzymes and into the expression bacterial was using The construct DdCBM8 to an tag with a site for for tag Recombinant DdCBM8 was in E. coli A was used to with as a was at of at by with for at by were in binding 5 and and on with for were and the were with for at The were with of and and the proteins were with tag with the protein was with a with and DdCBM8 was at The pull-down was as F. Y. S. T. carbohydrate-binding module of from a 2002; PubMed Scopus Google Scholar), with Ten of protein were in of or in for at and The was at for and the soluble was and with The insoluble was with and 1 the was in and insoluble were using was using a of a novel family of carbohydrate-binding modules with broad ligand Rep. 2016; 6: PubMed Scopus Google Scholar, Boraston A.B. Gilkes N.R. Kilburn D.G. of binding of the family carbohydrate-binding module from to and of amino acid 13: Google Scholar). and of protein were for 2 at and were at at for 5 and the protein of the was using the from was as B. S. S. M.A. Molecular basis for the and specificity of ligand recognition by the family carbohydrate-binding modules from Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, Kilburn D.G. of polysaccharides with the domain of specificity and analysis PubMed Scopus Google Scholar). were of of the soluble glucomannan, and The were using in the of was from the and proteins were at to the In all DdCBM8 was in the gel with or soluble polysaccharides. was used as the was at at for 2 were by with were using a using a with DdCBM8 in at The was and the were the of to were for The were for by and carbohydrates, for and for and all the were in binding were by of the The binding and the number of binding sites were using the and are the in the and of the ligand is the ligand In this is by the and the number of The of the carbohydrates are an by the and DdCBM8 was to and to with the of a from the at the The of protein and of were in and at and were used as the The first to the of the using the 2 of protein and 2 of and were at The of DdCBM8 were in in and and in a of at K. a DdCBM8 was in The were at the of the using a were with L. O. a new for with Biol. PubMed Scopus Google Scholar) and W. Biol. PubMed Scopus Google Scholar) and with and of Biol. 2006; PubMed Scopus Google Scholar). were using the using the A.J. in structure using of The Biol. PubMed Scopus Google Scholar) from G. Chen N. G.J. A.J. R. A for structure Biol. PubMed Scopus Google Scholar). The from a were identified by molecular using A.J. crystallographic 2007; PubMed Scopus Google Scholar) with as the The were using G. Chen N. G.J. A.J. R. A for structure Biol. PubMed Scopus Google Scholar) and with in B. K. and of Biol. PubMed Scopus Google Scholar). were deposited in the with and to and was using the The was used as a and the the were as and and and and and and and The proteins were and as in the on CBM8 and families in the Y. X. J. Chen X. F. Y. a for carbohydrate-active enzyme Acids Res. PubMed Scopus Google Scholar), the amino acid sequences of the domains were from and with a of sequence were using M.A. G. R. H. F. A. R. Thompson J.D. D.G. and 2007; 23: PubMed Scopus Google Scholar) and the The was using A and for Acids Scholar) based on the conserved amino acid soluble polysaccharides and were from for and which were from The bacterial cellulose production has been (22Campos B.M. Liberato M.V. Alvarez T.M. Zanphorlin L.M. Ematsu G.C. Barud H. Polikarpov I. Ruller R. Gilbert H.J. De Mattos Zeri A.C. Squina F.M. A novel carbohydrate-binding module from sugar cane soil metagenome featuring unique structural and carbohydrate affinity properties ∗.J. Biol. Chem. 2016; 291: 23734-23743Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, Barud M. M. Y. from bacterial cellulose and based as substrates for Chem. C. 2015; 3: Google Scholar). The that the of this are from the This The that have no of interest with the of this We the of on the and at the for in and M. V. B. M. and F. M. S. M. V. B. M. G. L. I. W. A. C.M. N. and F. M. S. F. M. S. M. V. G. and W. G. M. V. G. W. N. and F. M. S. F. M. S. 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