Xyloglucan Is Recognized by Carbohydrate-binding Modules That Interact with β-Glucan Chains

Abstract

Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of β-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a β-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated β-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains. Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of β-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a β-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated β-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains. The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has considerable industrial potential (1Coughlan M.P. Biotechnol. & Genet. Eng. Rev. 1985; 3: 39-109Crossref Scopus (289) Google Scholar). The complex chemical and physical structure of the plant cell wall restricts enzyme access to the polysaccharides, primarily cellulose and hemicellulose, that comprise this composite macromolecule. Microbial cellulases and hemicellulases generally contain noncatalytic carbohydrate-binding modules (CBMs) 4The abbreviations used are: CBM, carbohydrate-binding module; PKD, polycystic kidney disease; HEC, hydroxyethylcellulose; CMC, carboxymethylcellulose; AGE, affinity gel electrophoresis; ITC, isothermal titration calorimetry; BSA, bovine serum albumin; CAPS, 3-(cyclohexylamino)propanesulfonic acid; GH, glycoside hydrolase; CfCBM4-1, C. fimi CBM4-1; PeCBM29-2, P. equii CBM29-2; CtCBM11, C. thermocellum CBM11; CcCBM17, C. cellulovorans CBM17; BspCBM28, Bacillus spp. CBM28; SeMet, seleno-methionine; MAD, multi-wavelength anomalous dispersion. that target these enzymes to specific sites within the plant cell wall (2Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1532) Google Scholar). CBMs potentiate the hydrolytic activity of these biocatalysts by bringing the appended catalytic modules into intimate contact with their target substrates, thereby reducing the “accessibility problem” (2Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1532) Google D.N. P. M.P. Gilbert H.J. Biochem. J. PubMed Scopus Google J. Bolam D.N. M.P. Gilbert H.J. Biochem. J. PubMed Scopus Google Scholar). on structure CBMs have been into Gilbert Davies in of Scholar). studies on of the of CBM that these modules display a β-sandwich which has to the of a CBM (2Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1532) Google Scholar). of of The of CBM which in proteins that are of the the conformation of the target in which with the of polysaccharides, the binding of a hydrophobic platform that contains three aromatic J. J. PubMed Scopus Google Gilbert H.J. M.P. PubMed Scopus Google J. J. PubMed Scopus Google Scholar). CBMs affinity polysaccharides, and the ligand of CBM that contain modules is In which bind to single polysaccharide chains, accommodate the within of PubMed Scopus Google Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google A.B. Davies G.J. PubMed Scopus Google Bolam D.N. Gilbert H.J. Davies G.J. J. PubMed Scopus Google Scholar). In these polysaccharide is within of these and the catalytic of their catalytic CBM display in their to accommodate both in the that the backbone and the and of the in ligand is in CBM which contains proteins that recognize cellulose glucose glucose and and such as Bolam D.N. Gilbert H.J. J. 2004; PubMed Scopus Google Bolam D.N. J. Gilbert H.J. J. PubMed Scopus Google C. Gilbert H.J. A.B. J. PubMed Scopus Google Scholar). In addition, in ligand in a single polar residues in are able to to the in and the in glucose the to bind and Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google Scholar). studies on CBMs that recognize the and that the side chains of decorated are and not ligand binding Bolam D.N. Gilbert H.J. J. 2004; PubMed Scopus Google Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google J. Bolam D.N. M.P. C. Davies G.J. Gilbert H.J. J. PubMed Scopus Google A.B. P. J. PubMed Scopus Google is that this feature of CBMs these proteins to with to decorated and The of this to with to the key that contain a backbone of glucose that can by contain side chains such as xyloglucan. these are in plant cell in the cell wall of and in CBMs can display affinity β-1,4-glucans such as xyloglucan, and to the that this binding we have the structure and of the and C-terminal of CtCel9D-Cel44A Clostridium which is a component of the J. PubMed Google Scholar). enzyme contains glycoside and catalytic in to a and a C-terminal of Here we that the C-terminal domain of CtCel9D-Cel44A comprises a novel CBM (designated CBM44) that affinity xyloglucan. The structures of and with that both modules contain a single ligand cleft with a of three aromatic residues that are displayed to the of the The mechanism by which CBMs that recognize cellulose are also able to accommodate the side chains of xyloglucan is and is a modular enzyme by catalytic to and a and a C-terminal domain of The of the C-terminal domain of CtCel9D-Cel44A with polycystic kidney in a of and and by C. thermocellum the in and into and to and The to that the that used the The which contain a C-terminal in the and in to to a of and the a The proteins by affinity as Davies G.J. Gilbert H.J. J. 2004; PubMed Scopus Google Scholar). of to as by Bolam D.N. Gilbert H.J. J. 2004; PubMed Scopus Google and the by the the that the also and by affinity the proteins into and to gel by a with in and and three with the the fimi C. thermocellum Clostridium cellulovorans Bacillus spp. and equii as Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google and Bolam D.N. Gilbert H.J. J. 2004; PubMed Scopus Google J. P. PubMed Scopus Google A.B. P. PubMed Scopus Google A.B. Biochem. J. PubMed Scopus Google of and HEC, which and which and cellulose as Scopus Google Scholar). affinity of and CBM44 a of by affinity gel The as by P. Enzyme PubMed Scopus Google the polysaccharide a of in The bovine serum of binding as Scopus Google polysaccharide to of and CBM44 binding to and cellulose as of in and with of ligand in a of The with which the ligand by The comprising the and the three with of The bound by the in of and by a polysaccharide in to that of the CBM the J. Davies G.J. Gilbert H.J. Bolam D.N. J. 2004; PubMed Scopus Google a titration and the ligand in the to the of a titration the in a cell with a single by of ligand comprising polysaccharide of by a single binding The of sites in the polysaccharide as D.N. Gilbert H.J. J. 2004; PubMed Scopus Google Scholar). The the and the of binding the of and CBM44 the to the and as the The of the used to these are displayed in The to that only the been into the Enzyme activity of of CtCel9D-Cel44A as Davies G.J. Gilbert H.J. J. 2004; PubMed Scopus Google by the of of reducing the J. Google Scholar). in the of and CAPS, used in the enzyme The of the confirmed by the of reducing three are the of three and both and the crystal crystal and of by the with equal of in and and which a of by of in the a and in The used to are PubMed Scopus Google Scholar). on a with with a and the on the the a with the a to the The the of one in the and a of J. PubMed Scopus Google Scholar). of with equal of in and and in which a of a in a and in a crystal and the showed that to the with cell a and of a single crystal the a a fixed of the crystal the of the in the and that the crystal to the of the of in the with a of J. PubMed Scopus Google Scholar). the and J. 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PubMed Google a CBM and a C-terminal domain of which affinity J. PubMed Scopus Google a pivotal role in the of a as activity J. PubMed Google Scholar). The of the C-terminal of CtCel9D-Cel44A comprises a of this is discussed the display with other modules in glycoside The in this that this C-terminal of CtCel9D-Cel44A binds to plant and is as a novel CBM (designated the of CBM of CBM44 with CBM and the of three the and of these in CBM44 and in other CBM play a key role in the of a the of CBM that display a β-sandwich into a (2Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1532) Google to the of glycoside Although the these CBM as ligand and the of the residues can within a of and and to and their that both proteins displayed affinity xyloglucan, HEC, and a and binding to that the CBM with the of both proteins to with the cellulose and cellulose not The of CBM44 and the that the domain not to recognition. CBM44 is a CBM, binding β-1,4-glucan chains both in and that a ligand to the affinity are the of and CBM44 by affinity gel are as the ligand The are as the ligand in a of and CBM44 by binding of and CBM44 to their also by The in and that CBM44 binds to xyloglucan, HEC, and with xyloglucan is polysaccharide The that both and CBM44 not with that the of with the one The affinity of both and CBM44 the of a the of glucose with the is also that the orientation of the aromatic platform in the binding that adopt the conformation displayed by the xylose polymer J. and of of CBM44 and and as by of binding sites on the are are is affinity to of binding sites on the are J. PubMed Scopus Google is affinity to in a of CBM44 with showed that the affinity binds with to and The of binding is that CBM44 has a single The of both and CBM44 with and is with to ligand of is of the binding of CBMs to and the these has been discussed A.B. Davies G.J. PubMed Scopus Google Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google A.B. P. J. PubMed Scopus Google Bolam D.N. M.P. Gilbert H.J. PubMed Scopus Google Scholar). The that and the xyloglucan of is a with xylose and is bind to CBM44 with is with the polysaccharide that the side chains in xyloglucan not with the CBM also not to binding and not The that and CBM44 bind to xyloglucan the that CBMs are able to accommodate the side chains of this decorated The of the CBMs is with the of the appended studies on CBMs have not their affinity xyloglucan, we the to bind this polysaccharide is a feature of CBMs that recognize single chains. We used to the affinity of CfCBM4-1, CtCBM11, CcCBM17, BspCBM28, and bind to xyloglucan. The that five modules recognize xyloglucan. is that the xyloglucan side chains with the of CtCBM11, CcCBM17, and these modules bind to the decorated which the The affinity of xyloglucan and that the side chains of the decorated not with the The in affinity of xyloglucan, with that the xylose side chains are a to that to the the binding and the in affinity xyloglucan not to the of the to recognize the xylose side chains Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google of CBMs xyloglucan and as by in a of the crystal structures of A.B. Davies G.J. J. Scopus Google A.B. P. J. PubMed Scopus Google and Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google in complex with cello-oligosaccharides into the mechanism by which these proteins bind xyloglucan. 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J. 2004; 382: 769-781Crossref PubMed Scopus (1532) Google Scholar). the of a of enzymes to the located in plant cell in CBM44 would to target substrates, the and cell wall in the of a of other this the catalytic of both and xyloglucan and in the and of that not as this The in that CBM44 the activity of in the of the and not as can with the of the we that in the of the plant cell wall, CBM44 the activity of by the enzyme to the of this structure are of structure of with a The of contains and The structure the of and both a β-sandwich located in the has the side of and and the of with the with a single the in also and is bound to residues and and and The the and the to In β-sandwich CBMs have one which is and is to the (2Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1532) Google Scholar). The of in the of a CBM by that of the the by this on ligand binding P. M.P. Gilbert H.J. Bolam D.N. PubMed Scopus Google Scholar). has been that in a and a CBM a role in ligand Bolam D.N. Gilbert H.J. M.P. J. PubMed Scopus Google A.B. Davies G.J. 2004; PubMed Scopus Google Scholar). The sites in the domain and CBM44 are the structures in both a role the is to that CBM44 and which play a key role in are residues to the and and are in of the β-sandwich CBM the other residues to these are in the of the the that to the and is in the β-sandwich the that these three a role of the CBM by the we that the of β-sandwich CBMs a that is on the face of the contain these three The domain is with three and face to face and of a β-sandwich The has a concave The and of these are to of the and and the domain can as β-sandwich J. C. J. PubMed Scopus Google Scholar). The of the is three and one these residues are in the domain not are of this in the of a cell by the which is in polycystic kidney J. C. J. 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Bolam D.N. M.P. C. Davies G.J. Gilbert H.J. J. PubMed Scopus Google Bolam D.N. Gilbert H.J. J. 2004; PubMed Scopus Google M.P. C. Gilbert H.J. Bolam D.N. PubMed Scopus Google Scholar). In is that a role in ligand binding the other aromatic residues, which is with the that and are proteins that contain in is not In located in a to one of the binding in ligand the other tryptophan residues in the cleft J. Bolam D.N. M.P. C. Davies G.J. Gilbert H.J. J. PubMed Scopus Google that the hydrophobic with the and we a the of ligand in the of not contain the the binding residues are and is that these proteins not a carbohydrate-binding In CBM44 the of the aromatic in the binding ligand that the located on the concave of the comprises the and In contrast to of the tryptophan residues that and a in affinity and and is that a hydrophobic with the the the of this also the of and the the aromatic in the ligand binding of the of C. fimi also a role in cellulose binding the A.B. Eng. PubMed Google that the of this and cellulose a role in ligand of CBM44 xyloglucan as by binding binding in a The that the hydrophobic platform by the three tryptophan residues in both and CBM44 play a pivotal role in ligand binding The orientation of these residues in CBM44 is such that bind a twisted with the conformation by cello-oligosaccharides in and bound to a of CBMs Bolam D.N. Gilbert H.J. Davies G.J. PubMed Scopus Google A.B. P. J. PubMed Scopus Google A.B. Davies G.J. J. Scopus Google Scholar). The of the hydrophobic platform in CBM44 is and the of the aromatic residues would binding to and which that the can accommodate is to that CBM44 affinity is that residues of the hydrophobic platform of CBM44 with studies that and which are to and not to ligand binding and and C. fimi affinity P. PubMed Scopus Google the only with three glucose residues A.B. Davies G.J. J. Scopus Google Scholar). is that the by these the conformation by the in the binding the not with of the reducing the by this binding and these CBMs bind to of chains. In the orientation of the aromatic residues is is the and bind the face of the and in that a twisted the binds to the is to the hydrophobic The mechanism the binding of that the hydrophobic platform to CfCBM4-1, to the role in ligand of residues in CBM44 that are and in to the hydrophobic which a key role in ligand as In these only a in as by and showed that the of the the five xyloglucan, HEC, and not that these residues not with specific is that the of the proteins and are not the are to with and which are the three aromatic residues. the of a in affinity a that is to to the that the aromatic Although polar residues not play role in ligand in CBMs A.B. Eng. PubMed Google that bind to to the binding of CBMs to their target Bolam D.N. Gilbert H.J. J. 2004; PubMed Scopus Google M.P. C. Gilbert H.J. Bolam D.N. 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PubMed Scopus Google Scholar). such CBM44 is is that the not with and that is primarily the three aromatic residues in the binding the side chains in xyloglucan not the binding of the polysaccharide to as would the xylose and that with the The plant cell wall is structure by a complex of in which is a of cellulases that the enzyme to in a of xyloglucan, and Here we that of CtCel9D-Cel44A is a that is of a of plant cell wall such as and activity is that the role of a domain of is with the of to substrates, which are in a complex polysaccharide mediate ligand by CBM44 to the of and we that this in is to The of CBM44 are to the binding are the of CBMs the β-sandwich the crystal structures of and CBM44 that the in the structure the studies that the aromatic residues that comprise the hydrophobic platform in the of and CBM44 play a pivotal role in ligand is that the of the three binding residues on the of the cleft and CBM44 to bind β-1,4-glucan in to such as The of the binding and the of polar residues in ligand in that the in to the in ligand displayed by and the biological of CBMs that recognize single β-1,4-glucan chains is to target xyloglucan the of cellulose is this the of a major in the of this In a action CBM44 and target the of and that are in intimate contact with these are both catalytic as on cellulose and as on the of the to the and plant cell wall