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J. Biol. Chem., Vol. 279, Issue 20, 21560-21568, May 14, 2004
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From the
CIISA-Faculadade de Medicina Veterinaria, Rua Prof. Cid dos Santos, 1300-477 Lisboa, Portugal, Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE17RU, United Kingdom, ¶Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, Unite Mixte de Recherche 6098, CNRS Marseille and University Aix Marseille I & II, 31 chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France, and ||Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain
Received for publication, February 13, 2004 , and in revised form, March 5, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-linked oligosaccharides stack against Trp-92 and Tyr-33, whereas the rest of the binding cleft is blocked by Glu-20 and Thr-23, residues that are not present in CBM6 proteins that bind to the internal regions of polysaccharides in cleft A. Cleft B is solvent-exposed and, therefore, able to bind ligands because the loop, which occludes this region in other CBM6 proteins, is much shorter and flexible (lacks a conserved proline) in CmCBM6. Subsites 2 and 3 of cleft B accommodate cellobiose (Glc--1,4-Glc), subsite 4 will bind only to a -1,3-linked glucose, whereas subsite 1 can interact with either a -1,3- or -1,4-linked glucose. These different specificities of the subsites explain how cleft B can accommodate -1,4--1,3- or -1,3--1,4-linked gluco-configured ligands. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-strands (Ref. 2; see also afmb.cnrs-mrs.fr/CAZY).
The specificity of CBMs varies between families. All characterized proteins in CBM1, -5, and -10 bind to crystalline cellulose (4, 5), whereas other families contain modules that bind to a variety of different ligands, exemplified by CBM4, which contains proteins that recognize cellulose, xylan, mannan, and laminarin (6-9). The CBM6 family contains modules from 
35 enzymes (10) that display a diversity of substrate specificities. CBM6 proteins that bind to cellulose, xylan, mixed -(1,3)(1,4)glucan and -1,3-glucan have been described, and CBM6 sequences are also present in a 
-1,6-mannanase (Swiss-Prot accession number Q9Z4P9) and several -agarases (GenPept accession number AAF26838
[GenBank]
. The three-dimensional structure of two CBM6 modules from Clostridium thermocellum xylanase 10A (CtCBM6) (11) and a putative Clostridium stercorarium xylanase (CsCBM6) (12) have been determined. In contrast to all other CBM families, the CBM6 modules contain two clefts that could potentially function as ligand binding sites. Cleft B is located on the concave surface of one -sheet, whereas cleft A is found in the loop region connecting the inner and outer -sheets of the jelly roll fold. Cleft B is in a similar location to the ligand binding sites of CBMs from several other families that include CBM22, CBM4, CBM29, and CBM17 (13-16), whereas cleft A is positioned in the loops connecting the two -sheets and resembles the sugar binding sites of lectins, as has been discussed by Boraston et al. (12). NMR and mutagenesis studies on CtCBM6 and the resolution of the structure of CsCBM6 in complex with xylotriose revealed that in both proteins the xylo-configured ligands bind exclusively to cleft A; however, it was suggested that cleft B may play a role in carbohydrate recognition in family 6 proteins that display specificities distinct from CtCBM6 and CsCBM6. To address this issue we have analyzed the structural basis for the ligand specificity displayed by the C-terminal CBM6 (CmCBM6-2) of endoglucanase 5A from Cellvibrio mixtus. The accompanying paper (29) shows that CmCBM6-2 can accommodate 1,4- and 1,3-linked glucans in cleft A and B, and indeed, the two binding sites are required to bind insoluble cellulose. Only cleft B, however, interacts with (1,4)(1,3)-mixed linked glucans such as lichenan and barley -glucan, supporting the hypothesis that variation in ligand specificity within the CBM6 family reflects the functionality of the two potential carbohydrate binding clefts. Here we define the structural determinants for the distinct ligand specificity displayed by cleft A and cleft B in CmCBM6-2. Furthermore, by solving the structure of CtCBM6 with a xylooligosaccharide that occupies the complete binding site, we provide insight into the mechanism by which cleft A in family 6 CBMs can distinguish between gluco- and xylo-configured ligands.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-(1,4)(1,3)-linked glucan (Glc-4Glc-3Glc-4Glc-(1)-OMe, see Fig. 1; in all cases the ligand concentrations were 2 mM) yielded crystals in 11% polyethylene glycol 6000 and 2.0 M NaCl, whereas the complexes of CmCBM6-2 with two mixed -(1,3)(1,4)-linked glucans (Glc-3Glc-4Glc-3Glc and Glc-4Glc-3Glc-4Glc-OMe; Fig. 1), cellobiose and cellotriose, were obtained by soaking experiments. In the latter case native crystals were transferred into 20-µl drops of mother liquor containing 10% glycerol and 2-3 mM of oligosaccharides. After 20-30 min of soaking the crystals were mounted in a cryo loop and frozen in a 100 K nitrogen stream.
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2mM oligosaccharide) and 2 µl of reservoir solution containing 20% polyethylene glycol 1000, 0.2 M sodium acetate, and 0.1 M acetate buffer at pH 4.6. The crystals grew after 10 months at 4 °C. Here, the crystals belonged to space group P21 with unit cell parameters a = 27.48 Å, b = 51.68 Å, c = 36.13, and = 102.78°. The Vm value was calculated to be 1.9 Å3/Da with 1 molecule in the asymmetric unit. Data Collection, Structure Determination, and Refinement—All crystals were transferred to a cryoprotectant solution comprised of the growth buffer supplemented with 15% (v/v) glycerol. Subsequently the crystals, mounted in cryo loops, were flash-frozen in a cold nitrogen stream at 100 K. The different complex data sets were collected at beamlines ID29, ID14-EH1, and ID14-EH3 at the European Synchrotron Radiation Facilities, Grenoble, France. The details of data collection statistics are resumed in Table I. The data were processed and reduced with MOSFLM (17), and all further computing used the CCP4 (18) suite unless otherwise stated. The crystal structure of CmCBM6-2 was solved by the molecular replacement method using the program AMoRe (19) and the coordinates of the CsCBM6 (Protein Data Bank code 1nae [PDB] ) as the molecular starting model. Two solutions, corresponding to the two molecules in the asymmetric unit, gave a correlation coefficient of 22% and an R-factor of 53%. The model building was performed with ARP/wARP (20) using the complete data set at 1.40 Å. Then the structure was refined with REFMAC5 (18) to a final R-factor of 16.07% and Rfree factor of 17.76%. All complex structures having the same space group as the native protein were directly submitted to a first refinement cycle with REFMAC5 before manually constructing the substrate molecules using the program TURBO (21) and adding the water molecules. The same set of reflections was flagged for the calculation of the free R value for these complexes. Because the co-crystals of the CtCBM6 with xylopentaose did not have the same space group as the native crystals, molecular replacement with AMoRe was applied to position the protein molecules correctly in the new unit cell. The same strategy was applied to solve the structure of the crystals of CmCBM6-2 in complex with Glc-4Glc-3Glc-4Glc-OMe. All structures were refined with REFMAC5. The final refinement statistics are summarized in Table I. All coordinates of the different structures have been deposited with the Protein Data Bank with accession codes as follows: CtCBM6 with xylopentaose, 1uxx [PDB] ; native CmCBM6-2, 1uxz [PDB] ; CmCBM6-2 with cellobiose/cellotriose, 1uxy [PDB] /1uyy; CmCBM6-2 with Glc-4Glc-3Glc-4Glc-OMe, 1uz0 [PDB] ; CmCBM6-2 with Glc-3Glc-4Glc-3Glc, 1uy0 [PDB] ; CmCBM6-2 with xylotetraose, 1uyz [PDB] .
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-jelly roll fold, consisting predominantly of two five-stranded -sheets (Fig. 2). The overall structure is very similar to the two xylan binding family 6 CBMs previously described, with a root mean square deviation value of 0.9 Å over 123 matched C atoms with CsCBM6-3 and a value of 1.1 Å over 125 matched C atoms with CtCBM6.
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-sheets, analogous to the location of the binding clefts of CBM4, CBM15, CBM17, CBM22, and CBM29 (13-16, 22). Data presented below and in the accompanying paper (29) demonstrate that cleft B does indeed comprise a ligand binding site in CmCBM6-2 and that Trp-39 plays a pivotal role in carbohydrate recognition. By contrast, cleft B in both CsCBM6-3 and CtCBM6 is occluded from solvent by a proline residue in the neighboring surface loop and, thus, is not accessible to oligosaccharide or polysaccharide ligands. Furthermore CsCBM6-3 does not contain an aromatic residue in the equivalent position to Trp-39, which would further limit the capacity of cleft B in the C. stercorarium protein to bind ligand.
The Complex of CtCBM6 with Xylopentaose—Previously, we have shown that CtCBM6 binds with high affinity to xylans (unsubstituted and highly decorated) and xylooligosaccharides displaying the highest affinity for ligands with a degree of polymerization 
5, suggesting the presence of five sugar binding sites (11).
The structure of CtCBM6 in complex with xylopentaose was determined to a 1.6-Å resolution (see "Experimental Procedures" for details). Electron density for all five sugar units in xylopentaose was well defined, allowing unambiguous modeling of the pentasaccharide bound to cleft A (Fig. 3A). The direction of the sugar in the binding cleft was resolved by assessing the temperature factors and the level of electron density of the sugar C5 and O5 atoms and the hydrogen bonding of O5. Xylopentaose binds in an almost 3-fold helical orientation characterized by the internal hydrogen bond from O3n to O5n+1. The chair ring planes (defined by C2, C3, C5, and O5) of the three sugars Xyl2-4 (Xyl1 defines the reducing end of the ligand, and Xyl5 defines the non-reducing end) lie at angles of 89 and 84°, respectively. Xylan thus binds in its favored 3-fold helical conformation previously determined by x-ray fiber diffraction analysis of xylan (23). Such a conformation has previously been proposed for xylohexaose bound to CBM2b-1 from Cellulomonas fimi Xyn11A (24), and observed when the ligand is bound to CBM15 from Cellvibrio japonicus Xyn10C (22).
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-sheets that form the sandwich (22, 26). In contrast, the binding site both in CtCBM6 (11) and CsCBM6-3 (12) is unusual; it is not located in the central groove, which contains the ligand binding site in most of the CBMs studied to date but instead is on one edge of the sandwich. This binding site, designated "cleft A," from CsCBM6-3 binds to oligosaccharides derived from xylan and cellulose with similar affinities (12), whereas in CtCBM6 cleft A accommodates primarily xylan and xylooligosaccharides (11). The binding site (cleft A) is formed by two loops, residues 28-34 and 90-97, which contain two highly conserved aromatic residues, Tyr-33 and Trp-92, that stack against the and face of the central xylose of the pentasaccharide located in subsite 3, whereas Asn-120 forms a tight hydrogen bond (2.6 Å) with O3 of this sugar. It is interesting to note that although two aromatic residues are also involved in ligand binding in the xylan binding CBMs from family 2a and 15, these residues are in a perpendicular orientation with respect to each other and, by stacking against xylose residues n and n + 2, play a key role in defining a binding site topology that can accommodate ligands with a 3-fold helical conformation (22, 24). Further subsites are formed in CtCBM6 by Asp-64 and Thr-65, which bind to the reducing end of the ligand at subsite 1, Pro-118 is at subsite 2, Ile-23 and Asn-93 form subsite 4, whereas Gly-24, Ser-26 (weakly; OG 3.9 Å and N 3.8 Å), and Asn-93 interact with the non-reducing end of xylopentaose at subsite 5 (Fig. 3, B and C). The distances of hydrogen bonds and close contact are summarized in Table II. On the basis of NMR data we had speculated that the surface loop from Gly-24 to Gly-33 might undergo a conformational change upon binding (11), and Boraston et al. (12) observed that Phe-45 (Ile-23 in CtCBM6) in the equivalent loop in CsCBM6-3 moves by 5 Å when bound to xylotriose. Here we show that residues 25-27 undergo a positional shift, bringing Ser-26 (shifted by 4.1 Å) closer to the sugar at subsite 1 and, thus, facilitating the weak interaction between the hydroxy amino acid and O4 of the xylose at subsite 5. Although such conformational changes are likely to incur an energetic cost, which will lead to a reduction in overall affinity, the movement of the protein may reflect the heterogeneity of the xylan decorations. Thus, although the movement of Ser-26 is optimal for binding unsubstituted xylooligosaccharides, this amino acid may need to be more distant from the binding cleft to accommodate xylans that are decorated at O2 and/or O3.
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Comparison of the CtCBM6-xylopentaose complex with that of CsCBM6-3 bound to xylotriose at subsites 2, 3, and 4 reveals a similar arrangement of residues and surface loops around these three central subsites (Fig. 4). By contrast, residues involved in ligand binding at subsites 1 and 5 in the CtCBM6-xylopentaose complex have no counterpart in CsCBM6-3. In the region forming subsite 1 of CtCBM6, there is a 1-residue insert in the equivalent region of CsCBM6-3, and the backbone positions of the residues cannot be superimposed. As a consequence, Asp-64 and Thr-65 have no equivalent residues in CsCBM6-3; however, the ND2 of Asn-85 in the C. stercorarium protein is predicted to make a hydrogen bond (2.9 Å) with O3 of the xylose at subsite 1. A similar situation is observed in the region forming subsite 5. Here, the positions of the loops formed by residues 22-32 in CtCBM6-3 and 44-54 in CsCBM6-3 are different. CsCBM6-3, however, contains several residues that are likely to form a functional subsite 5; Pro-48 is predicted to stack again the xylopyranose ring, whereas Asp-113 OD1 and the backbone carbonyl of Ser-46 will make hydrogen bonds with O5 and O2, respectively. Thus, both CsCBM6-3 and CtCBM6 are likely to contain five subsites, although the structures of the distal subsites in the two proteins are very different.
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1,4-1,3-mixed linked glucans lichenan, and barley -glucan, insoluble forms of cellulose and displays weak affinity for the 1,3-glucan laminarin. Additionally, mutagenesis studies show that both cleft A and B can accommodate laminarin and cellulooligosaccharides, whereas only cleft B binds to glucans with mixed -1,4--1,3 linkages such as lichenan and barley -glucan. It also appears that the binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B (see the accompanying paper, Ref. 29).
We were able to obtain complexes of CmCBM6-2 with five different ligands, cellobiose, cellotriose, xylotetraose, and two 1,4-1,3-mixed linked glucans (Glc-4Glc-3Glc-4Glc-OMe and Glc-3Glc-4Glc-3Glc; see Fig. 1). However, no crystals of complexes with pure -1,3-linked glucans were obtained either by soaking or co-crystallization. The lack of complex formation for this substrate by soaking experiments might be due to crystal packing constraints since cleft A is in close contact to a neighboring molecule. The much lower affinity displayed by the two binding clefts for the -1,3-linked glucans might explain the lack of complex formation in the co-crystallization trials. The asymmetric unit for the cellulooligosaccharide complexes contains two protein molecules, so there are four putative ligand binding sites in the asymmetric unit (cleft A and B in both CmCBM6 molecules). In both the cellobiose and cellotriose complexes three of these sites are occupied, cleft A and B of the first molecule and only cleft B of the second molecule. Hereafter, ligand binding described for cleft A refers to one protein molecule in the asymmetric unit, whereas binding to cleft B relates to both copies of CmCBM6 in the asymmetric unit unless otherwise specified.
Three-dimensional Structure of CmCBM6-2 in Complex with Cellobiose and Cellotriose at Cleft A—In crystals of CmCBM6-2 soaked with cellobiose or cellotriose electron density compatible with the disaccharide was evident in subsites 3 and 4 (using the nomenclature for the subsites in CtCBM6 cleft A) of cleft A. The positions of C6 and O6 are undefined, which likely reflects ligand binding in both orientations. Electron density is only clearly defined for one sugar unit, the second sugar is disordered for both the di- and trisaccharide ligands, and no density is evident for the third sugar of cellotriose. The lack of electron density for a third sugar in the cellotriose complex suggests that one of the glucose units in this ligand is completely disordered, and thus, does not form stable interactions with cleft A. This is entirely consistent with the data of Henshaw et al. (29), which show that cleft A contains only two sugar binding subsites. The hydrogen bonds formed between protein residues and the hydroxyl groups of the sugar units as well as close contacts are summarized in Table III. Binding of cellobiose and cellotriose in cleft A occurs via stacking interactions of a terminal sugar unit between the two aromatic residues, Tyr-33 and Trp-92, at subsite 3, although the orientation of the bound ligand is unclear. In both orientations, three OH groups are stabilized by residues Glu-20 and Asn120 (Fig. 5 and Table III). For complexes in which the non-reducing end of the ligand is bound at subsite 3, O2, O3, and O4 interact with these two amino acids, whereas O1, O2, and O3 hydrogen bind to Glu-20 and Asn-120 when Glc1 (reducing end sugar) is sandwiched between the two aromatic residues in cleft A. The second glucose molecule interacts only with the hydroxyl group of Tyr-33 via either O2 or O3, depending on the orientation of the ligand, although the CBM does make indirect water-mediated hydrogen bonds with this sugar residue.
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-1,4-linked glucans with a degree of polymerization of 3 (29). Unlike cleft A, the electron density for both C6 and O6 in cellobiose and cellotriose are clear, indicating that these ligands are located in a single orientation in cleft B. Indeed the orientation of cellulose chains in CBM binding sites is a matter of some debate. The solution structure of a Cellulomonas CBM4 suggests that cellooligosaccharides can bind in both orientations (27), whereas the crystal structure of the same protein in complex with cellopentaose (15) and a CBM17 module bound to cellohexaose indicate that these ligands interact with the two CBMs in a single orientation (16). Uniquely, CmCBM6 appears to accommodate cellulooligosaccharides uni- or bi-directionally, dependent on the binding site occupied.
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Three-dimensional Structure of CmCBM6-2 in Complex with -1,4--1,3-linked Glucans—The crystal structure of Cm- CBM6-2 in complex with the mixed 1,4-1,3-linked ligand, Glc-4Glc-3Glc-4Glc-O-methyl, revealed that Glc4 binds in cleft A, whereas Glc1-Glc3 is located in cleft B of a second, crystallographically related CBM6 molecule (Fig. 7A). This reflects the capacity of cleft B to bind 1,4-1,3-linked glucans and cleft A to accommodate terminal glucose molecules in subsite 3; however, the stabilization of the trimolecular complex by ligand binding to 2 protein molecules most probably is an artifact of crystallization because cleft A does not contribute to the binding of mixed-linked glucans. Interestingly, Glc4 is bound in cleft A in a manner similar to Xyl1 of xylotetraose, with O1, O2, and O3 interacting with Glu-20 and Asn-121 (molecule A in Fig. 7A). The -1,4-linked Glc1 and Glc2 residues are bound in subsites 2 and 3 of cleft B, respectively, in a pattern identical to Glc2 and Glc3 of cellotriose (Fig. 6, A and B, and 7A). Subsite 4 is occupied by -1,3-linked Glc3, whereas subsite 1 is unoccupied. In subsite 4, three hydrogen bonds stabilize the glucose unit, Lys-114 with O6 and O5, whereas the carbonyl of Gly-37 interacts with O6. Subsite 4 is positioned relative to the two central subsites 2 and 3 such that only a -1,3-linked glucose can interact with this distal subsite. Furthermore, tight hydrogen bonds between subsite 3 and O6 and O4 prevents a -1,4-linked glucose moiety binding at subsite 4 (Fig. 7B).
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1,4-1,3-linked oligosaccharide Glc-3Glc-4Glc-3Glc the ligand is bound only in clefts B. In common with all other oligosaccharides located in cleft B the primary interaction between the protein and ligand is between subsites 2 and 3 and 2 -1,4-linked glucose moieties depicted in Fig. 6 and Table III. In one of the two molecules in the asymmetric unit only -1,4-linked Glc2 and Glc3 are defined by electron density, whereas in the second CmCBM6-2 molecule all four binding subsites in cleft B are occupied with glucose units. Thus, subsite 1 can accommodate a -1,3- or -1,4-linked glucose molecule; this latter, therefore, represents the maximum binding that can be obtained in cleft B with mixed -1,4-1,3-linked glucans.
Mechanisms of Ligand Recognition—The binding mode in cleft A is clearly different in CBM6 modules that display distinct specificities. The examples described here reflect the two extremes, CtCBM6, which is able to bind internal regions of xylan chains as reflected by its complex with xylopentaose (Fig. 3B), and CmCBM6-2, which binds to the terminal residues of both xylo- and gluco-configured oligosaccharides (Fig. 7a). In all CBM6 structures characterized to date, the conserved residues Asn-120, Trp-92, and Tyr-33 (numbers are from CmCBM6-2) play a pivotal role in ligand binding. These residues define the two subsites, 3 and 4, that are invariant in the three CBM6 proteins. CmCBM6-2 cleft A appears to bind only at subsites 3 and 4, whereas Boraston et al. (12) define 3 subsites in CsCBM6-3 and the complex of CtCBM6 with xylopentaose identifies 5 subsites. Overlaying the structure of CsCBM6-3 and CtCBM6 indicates that the C. stercorarium module also contains five subsites, although the structure of the distal subsites, 1 and 5, are very different in the two proteins. One of the key differences between CsCBM6-3 and CtCBM6 is that the C. thermocellum protein binds xylohexaose 100-fold tighter than cellohexaose, whereas the affinity of the C. stercorarium module for these two ligands is similar (xylohexaose binds 5 tighter than cellohexaose). The complex of CtCBM6 with xylopentaose provides some insight into the mechanism by which this protein is able to discriminate between xylo- and gluco- configured ligands. Specificity for xylose-containing polymers is conferred at subsites 4 and 5 where Ile-23 and Ser-26, respectively would make steric clashes with the C6OH of glucose. The capacity of CsCBM6-3 to bind cellohexaose more tightly than CtCBM6 may be due to differences in subsite 5, where the lack of an equivalent residue to Ser-26 in the C. stercorarium protein would facilitate the accommodation of the C6OH moiety of glucose. The accommodation of glucose in subsite 4 of CsCBM6-3 is less clear because Phe-45 (equivalent to CtCBM6 Ile23) would likely clash with the C6 group. It is possible that the cavity behind the aromatic side chain could provide sufficient space for Phe-45 to undergo a conformational change upon cellohexaose binding. Indeed, the 200,000-fold variation in the capacity of the -1 subsite of GH10 xylanases to accommodate glucose is dependent upon the conformational freedom of an invariant tryptophan at the active site; enzymes that contain a cavity behind the indole ring display the highest activity against gluco-configured substrates (28).
Inspection of the crystal structure of CmCBM6 provides some insight into its specificity for terminal sugar residues. The surface loop from Gln-18 to Tyr-33 adopts a slightly different conformation to the equivalent region of CtCBM6 and constricts the binding cleft at subsite 5. Indeed, overlaying the liganded CtCBM6 structure with CmCBM6 indicates that Thr-23 in the Cellvibrio protein will clash with O4 of the xylose at subsite 5. Furthermore, CmCBM6 Glu-20, the equivalent residue to Phe-45 and Ile-23 in CsCBM6-3 and CtCBM6, respectively, while playing a role in ligand binding at subsite 3, is predicted to clash with a sugar residue that occupies subsite 4.
CmCBM6-2 and CsCBM6-3 (12) are CBM6s for which binding has been observed in cleft B. From sequence alignments (see alignment in Boraston et al. (15) for example), however, one can conclude that several other CBM6 proteins will also have an equivalent ligand binding cleft. Our structure identifies a maximum of 4 binding sites in cleft B of CmCBM6-2, with subsites 2 and 3 comprising the major binding sites. Discrimination between gluco- and xylo-configured ligands occurs only at subsite 2, where the C6OH of glucose makes strong hydrogen bonds with Glu-73 and the backbone carbonyl of Glu-74, explaining why the protein does not bind xylan at cleft B. Supplementary binding is possible at subsites 1 and 4. At these distal binding sites two highly flexible residues, Lys-114 in subsite 4 and Gln-13 in subsite 1, undergo conformational changes so that the side chains can interact with glucose molecules located at these subsites. It is noteworthy that discrimination between -1,4-linked glucans (cellulose) and mixed -1,3-1,4-linked glucans occurs at subsite 4, where only a -1,3-linked glucose can be accommodated, whereas subsite 1 can interact with a sugar that is linked either -1,3 or -1,4. Thus, the order of the -1,3-1,4 linkages in glucans is important in defining affinity because only Glc--1,3-Glc--1,4-Glc--1,3-Glc and Glc--1,3-Glc-1,4-Glc--1,4-Glc will occupy all four subsites.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
** To whom correspondence should be addressed. Tel.: 33-491-164-513; Fax: 33-491-164-536; E-mail: czjzek{at}afmb.cnrs-mrs.fr.
1 The abbreviation used is: CBM, carbohydrate binding module.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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J. L. Henshaw, D. N. Bolam, V. M. R. Pires, M. Czjzek, B. Henrissat, L. M. A. Ferreira, C. M. G. A. Fontes, and H. J. Gilbert The Family 6 Carbohydrate Binding Module CmCBM6-2 Contains Two Ligand-binding Sites with Distinct Specificities J. Biol. Chem., May 14, 2004; 279(20): 21552 - 21559. [Abstract] [Full Text] [PDF]
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A-weighted 2 Fobs-Fcalc electron density map contoured at a 1 
