- Email: [email protected]
How Carbohydrate Binding Modules Overcome Ligand Complexity
Previews 609
berger, and coworkers show that the C terminus of Sir4 indeed forms a parallel coiled-coil structure (Chang et al., 2003). Although the coiled-coil structure conforms to the classical “knobs into holes” packing, amino acids occupying the a and d positions of the pseudo-heptad repeats deviate from a typical composition. Nevertheless, the coiled coil is stable, which unequivocally demonstrates that the full-length Sir4 forms a dimer via the coiled-coil interaction. The C-terminal region of Sir4 harbors the binding site of Sir3, but the precise location has not been determined (Moazed et al., 1997). Using the structural information, these investigators further show that the Sir3-interacting region is located in the coiled-coil domain of Sir4, and they pinpoint the critical contact area to a patch of hydrophobic residues at the center of the coiled-coil. They further mapped the reciprocal Sir4 interaction region on Sir3 to a short stretch of amino acids located in a flexible region connecting the N-terminal BAH-containing domain and the C-terminal defective AAA⫹ ATPase-like domain. Gel filtration, equilibrium sedimentation, and isothermal calorimetric experiments also show that the C-terminal domain of Sir3 forms a homodimer and that a Sir3 dimer interacts with a Sir4 dimer through the coiled-coil domain. In addition, the authors show that Sir2 binds to a separate region of Sir4 N-terminal to the coiled coil and that the binding of Sir2 is independent of that of Sir3. These studies not only provide direct confirmation of previous genetic and biochemical results, they also offer detailed information about protein-protein interactions governing the assembly of the Sir2/Sir3/Sir4 complex. These results will be of great value to guide further in-
depth genetic, biochemical, and structural dissection of the Sir2/Sir3/Sir4 complex during the establishment and maintenance of silent chromatin domains. Rui-Ming Xu W.M. Keck Structural Biology Laboratory Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724 Selected Reading Bryk, M., Banerjee, M., Murphy, M., Knudsen, K.E., Garfinkel, D.J., and Curcio, M.J. (1997). Genes Dev. 11, 255–269. Chang, J.F., Hall, B.E., Tanny, J.C., Moazed, D., Filman, D., and Ellenberger, T. (2003). Structure 11, this issue, 637–649. Ghidelli, S., Donze, D., Dhillon, N., and Kamakaka, R.T. (2001). EMBO J. 20, 4522–4535. Gottschling, D.E., Aparicio, O.M., Billington, B.L., and Zakian, V.A. (1990). Cell 63, 751–762. Hecht, A., Strahl-Bolsinger, S., and Grunstein, M. (1996). Nature 383, 92–96. Hoppe, G.J., Tanny, J.C., Rudner, A.D., Gerber, S.A., Danaie, S., Gygi, S.P., and Moazed, D. (2002). Mol. Cell. Biol. 22, 4167–4180. Moazed, D., Kistler, A., Axelrod, A, Rine, J., and Johnson, A. (1997). Proc. Natl. Acad. Sci. USA 94, 2186⫺2191. Moretti, P., Freeman, K., Coodly, L., and Shore, D. (1994). Genes Dev. 8, 2257–2269. Rine, J., and Herskowitz, I. (1987). Genetics 116, 9–22. Rusche, L.N., Kirchmaier, A.L., and Rine, J. (2003). Annu. Rev. Biochem. 72, 481⫺516. Smith, J.S., and Boeke, J.D. (1997). Genes Dev. 11, 241–254. Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997). Genes Dev. 11, 83–93. Renauld, H., Aparicio, O.M., Zierath, P.D., Billington, B.L., Chhablani, S.K., and Gottschling, D.E. (1993). Genes Dev. 7, 1133–1145.
Structure, Vol. 11, June, 2003, 2003 Elsevier Science Ltd. All rights reserved.
How Carbohydrate Binding Modules Overcome Ligand Complexity
The first crystal structure of a carbohydrate binding module in complex with a substituted oligosaccharide has provided important insights into how these proteins are able to target the backbone of complex polysaccharides that are extensively decorated. Protein-carbohydrate recognition plays a pivotal role in key biological processes. These macromolecular interactions are central to host-pathogen recognition, cellcell communication, cellular defense mechanisms, protein trafficking, and the recycling of photosynthetically fixed carbon through the degradation of the plant cell wall. Microbial enzymes that catalyze plant cell wall hydrolysis have a modular structure in which noncatalytic carbohydrate binding modules (CBMs) target the biocat-
DOI 10.1016/S0969-2126(03)00103-5
alysts to specific polysaccharides and enhance catalytic efficiency by increasing the effective concentration of the enzyme on the surface of insoluble substrates. CBMs are grouped into a number of discrete families based upon amino acid sequence similarity. Subtle differences in the structure of CBMs can lead to very diverse ligand specificity (Boraston et al., 2002; Simpson et al., 2000). Thus, CBMs in complex with their respective ligands present excellent systems for dissecting the molecular determinants that define the structural basis for protein-carbohydrate recognition. The three-dimensional structures of CBMs have shown that these protein modules are composed almost exclusively of  strands arranged in a “jelly roll” motif whose topography reflects the macroscopic nature of the target substrate. Modules that interact with crystalline cellulose, for example, display a planar hydrophobic surface that is thought to interact with adjacent chains on the surface of the crystal lattice (Tormo et al., 1996). Conversely, CBMs that bind amorphous cellulose, mannan, or xylan possess clefts, which interact with a single
Structure 610
chain of their poly/oligosaccharide ligands (Notenboom et al., 2001). One of the key issues with respect to CBMs is the heterogeneous nature of many of the target polysaccharide ligands; the backbone saccharide polymer is often decorated with an array of different sugars and acetate moieties, which vary in nature and extent dependent on the plant species and its differentiation state. How CBMs are able to bind to the backbone sugar polymer and yet display sufficient promiscuity in ligand recognition to accommodate these variable side chains is an important issue. Recent crystal structures of CBMs in complex with their target ligands have started to clarify how these proteins are able to bind to decorated ligands. Thus, the crystal structure of CBM15 in complex with xylopentaose showed that six of the ten C2-OH and C3-OH moieties were solvent exposed (Szabo et al., 2001), demonstrating how this protein was able to interact with xylose polymers that contained numerous side chains. Similarly, the structure of CBM29 bound to mannohexaose showed that the C6-OH groups of alternate mannose residues were solvent exposed, showing how this protein was able to interact with mannan that was decorated at C6 with galactosyl side chains (Charnock et al., 2002). The structures of CBM13 (Notenboom et al., 2002; Fujimoto et al., 2002) and CBM6 (Boraston et al., 2003a) bound to xylooligosaccharides also reveal information on the extent to which the side chains of xylans can be accommodated in the binding site of these proteins. The paper by Boraston et al. (2003b) presented in this issue of Structure makes a decisive contribution to understanding the mechanism by which CBMs are able to bind to decorated ligands. This group solved the structure of a novel family 27 mannan binding CBM in complex with mannohexaose and 63,64-␣-D-galactosylmannopentaose. The structure revealed how the protein is able to accommodate mannan, a 1,4-linked mannose polymer as opposed to cellulose, a 1,4-linked glucose polymer. The CBM makes numerous hydrogen bonds with the C2-OH of the mannose sugars, and predicted steric clashes between C2-OH equatorial sugar hydroxyls and the protein, which would preclude glucose from binding at subsites 3 and 4, provides an explanation for why this protein binds only to mannose-containing polymers. It is interesting to note that despite not binding to cellulooligosaccharides, or indeed accommodating glucose at two of the five subsites, the protein is able to bind the mixed glucose-mannose polymer glucomannan with essentially complete coverage. The Boraston paper importantly provides the first glimpses of a CBM bound to a decorated oligosaccharide. The galactosyl side chains can be accommodated in subsites 1, 2, and 5 where the C6-OH groups point into solvent. In subsite 4, the galactose side chains extend out from the edges of the ribbon formed by the 2-fold axis of the -1,4-linked mannose structure, consistent with the known structure of galactomannan. The galactose attached to the mannose in subsite 3, however, is located in a plane parallel to the mannose backbone. This galactose appears to be forced into this conformation by Trp-28, whose position would not allow the galactose side chain to extend away from the mannan backbone, and thus provides an explanation for the low
affinity toward the decorated ligand. These data demonstrate how CBM27 is able to bind a highly decorated galactomannan, as four of the five subsites are able to accommodate the sugar side chain. The Boraston paper also makes a significant contribution to our understanding of the stoichiometry of CBM ligand binding. The authors provide compelling biochemical evidence that at high protein concentration, two molecules of the CBM are bound to one molecule of mannohexaose. The oligosaccharide binds the two CBM molecules sequentially. The first binding event has a high affinity, whereas the second protein interacts only weakly with the CBM27-mannohexaose complex. The structural basis for this dimer-ligand complex is unclear, as no crystallographic data on this trimolecular complex are available. It is possible that the oligosaccharide spans the two CBMs and thus partially occupies the binding cleft of the two proteins. Such a binding event, however, would require a significant redistribution of the oligosaccharide that is not associated by a highly beneficial ⌬G. An alternative, more likely, hypothesis is that the second CBM molecule binds to the faces of the oligosaccharide that are not interacting with the already bound protein. Thus, even if this second CBM27-oligosaccharide interaction is weak, it does not compromise the initial high-affinity CBM27-mannohexaose binding event. Clearly, structural data are required to elucidate which mechanism is correct. The paper by Boraston et al. has made an important contribution to the mechanism by which CBMs are able to accommodate decorated polysaccharides, and provides new insights into the assembly of CBM-oligosaccharide complexes, and as such represents a landmark discovery in the field of carbohydrate protein recognition. Harry J. Gilbert School of Cell and Molecular Biosciences University of Newcastle upon Tyne Newcastle upon Tyne NE1 7RU United Kingdom Selected Reading Boraston, A.B., Nurizzo, D., Notenboom, V., Ducros, V., Rose, D.R., Kilburn, D.G., and Davies, G.J. (2002). J. Mol. Biol. 319, 1143–1156. Boraston, A.B., Notenboom, V., Warren, R.A., Kilburn, D.G., Rose, D.R., and Davies, G. (2003a). J. Mol. Biol. 327, 659–669. Boraston, A.B., Revett, T.J., Boraston, C.M., Nurizzo, D., and Davies, G.J. (2003b). Structure 11, this issue, 665–675. Charnock, S.J., Bolam, D.N., Nurizzo, D., Szabo, L., McKie, V.A., Gilbert, H.J., and Davies, G.J. (2002). Proc. Natl. Acad. Sci. USA 99, 14077–14082. Fujimoto, Z., Kuno, A., Kaneko, S., Kobayashi, H., Kusakabe, I., and Mizuno, H. (2002). J. Mol. Biol. 316, 65–78. Notenboom, V., Boraston, A.B., Chiu, P., Freelove, A.C., Kilburn, D.G., and Rose, D.R. (2001). J. Mol. Biol. 314, 797–806. Notenboom, V., Boraston, A.B., Williams, S.J., Kilburn, D.G., and Rose, D.R. (2002). Biochemistry 41, 4246–4254. Simpson, P.J., Xie, H., Bolam, D.N., Gilbert, H.J., and Williamson, M.P. (2000). J. Biol. Chem. 275, 41137–41142. Szabo, L., Jamal, S., Xie, H., Charnock, S.J., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2001). J. Biol. Chem. 276, 49061–49065. Tormo, J., Lamed, R., Chirino, A.J., Morag, E., Bayer, E.A., Shoham, Y., and Steitz, T.A. (1996). EMBO J. 15, 5739–5751.
berger, and coworkers show that the C terminus of Sir4 indeed forms a parallel coiled-coil structure (Chang et al., 2003). Although the coiled-coil structure conforms to the classical “knobs into holes” packing, amino acids occupying the a and d positions of the pseudo-heptad repeats deviate from a typical composition. Nevertheless, the coiled coil is stable, which unequivocally demonstrates that the full-length Sir4 forms a dimer via the coiled-coil interaction. The C-terminal region of Sir4 harbors the binding site of Sir3, but the precise location has not been determined (Moazed et al., 1997). Using the structural information, these investigators further show that the Sir3-interacting region is located in the coiled-coil domain of Sir4, and they pinpoint the critical contact area to a patch of hydrophobic residues at the center of the coiled-coil. They further mapped the reciprocal Sir4 interaction region on Sir3 to a short stretch of amino acids located in a flexible region connecting the N-terminal BAH-containing domain and the C-terminal defective AAA⫹ ATPase-like domain. Gel filtration, equilibrium sedimentation, and isothermal calorimetric experiments also show that the C-terminal domain of Sir3 forms a homodimer and that a Sir3 dimer interacts with a Sir4 dimer through the coiled-coil domain. In addition, the authors show that Sir2 binds to a separate region of Sir4 N-terminal to the coiled coil and that the binding of Sir2 is independent of that of Sir3. These studies not only provide direct confirmation of previous genetic and biochemical results, they also offer detailed information about protein-protein interactions governing the assembly of the Sir2/Sir3/Sir4 complex. These results will be of great value to guide further in-
depth genetic, biochemical, and structural dissection of the Sir2/Sir3/Sir4 complex during the establishment and maintenance of silent chromatin domains. Rui-Ming Xu W.M. Keck Structural Biology Laboratory Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724 Selected Reading Bryk, M., Banerjee, M., Murphy, M., Knudsen, K.E., Garfinkel, D.J., and Curcio, M.J. (1997). Genes Dev. 11, 255–269. Chang, J.F., Hall, B.E., Tanny, J.C., Moazed, D., Filman, D., and Ellenberger, T. (2003). Structure 11, this issue, 637–649. Ghidelli, S., Donze, D., Dhillon, N., and Kamakaka, R.T. (2001). EMBO J. 20, 4522–4535. Gottschling, D.E., Aparicio, O.M., Billington, B.L., and Zakian, V.A. (1990). Cell 63, 751–762. Hecht, A., Strahl-Bolsinger, S., and Grunstein, M. (1996). Nature 383, 92–96. Hoppe, G.J., Tanny, J.C., Rudner, A.D., Gerber, S.A., Danaie, S., Gygi, S.P., and Moazed, D. (2002). Mol. Cell. Biol. 22, 4167–4180. Moazed, D., Kistler, A., Axelrod, A, Rine, J., and Johnson, A. (1997). Proc. Natl. Acad. Sci. USA 94, 2186⫺2191. Moretti, P., Freeman, K., Coodly, L., and Shore, D. (1994). Genes Dev. 8, 2257–2269. Rine, J., and Herskowitz, I. (1987). Genetics 116, 9–22. Rusche, L.N., Kirchmaier, A.L., and Rine, J. (2003). Annu. Rev. Biochem. 72, 481⫺516. Smith, J.S., and Boeke, J.D. (1997). Genes Dev. 11, 241–254. Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997). Genes Dev. 11, 83–93. Renauld, H., Aparicio, O.M., Zierath, P.D., Billington, B.L., Chhablani, S.K., and Gottschling, D.E. (1993). Genes Dev. 7, 1133–1145.
Structure, Vol. 11, June, 2003, 2003 Elsevier Science Ltd. All rights reserved.
How Carbohydrate Binding Modules Overcome Ligand Complexity
The first crystal structure of a carbohydrate binding module in complex with a substituted oligosaccharide has provided important insights into how these proteins are able to target the backbone of complex polysaccharides that are extensively decorated. Protein-carbohydrate recognition plays a pivotal role in key biological processes. These macromolecular interactions are central to host-pathogen recognition, cellcell communication, cellular defense mechanisms, protein trafficking, and the recycling of photosynthetically fixed carbon through the degradation of the plant cell wall. Microbial enzymes that catalyze plant cell wall hydrolysis have a modular structure in which noncatalytic carbohydrate binding modules (CBMs) target the biocat-
DOI 10.1016/S0969-2126(03)00103-5
alysts to specific polysaccharides and enhance catalytic efficiency by increasing the effective concentration of the enzyme on the surface of insoluble substrates. CBMs are grouped into a number of discrete families based upon amino acid sequence similarity. Subtle differences in the structure of CBMs can lead to very diverse ligand specificity (Boraston et al., 2002; Simpson et al., 2000). Thus, CBMs in complex with their respective ligands present excellent systems for dissecting the molecular determinants that define the structural basis for protein-carbohydrate recognition. The three-dimensional structures of CBMs have shown that these protein modules are composed almost exclusively of  strands arranged in a “jelly roll” motif whose topography reflects the macroscopic nature of the target substrate. Modules that interact with crystalline cellulose, for example, display a planar hydrophobic surface that is thought to interact with adjacent chains on the surface of the crystal lattice (Tormo et al., 1996). Conversely, CBMs that bind amorphous cellulose, mannan, or xylan possess clefts, which interact with a single
Structure 610
chain of their poly/oligosaccharide ligands (Notenboom et al., 2001). One of the key issues with respect to CBMs is the heterogeneous nature of many of the target polysaccharide ligands; the backbone saccharide polymer is often decorated with an array of different sugars and acetate moieties, which vary in nature and extent dependent on the plant species and its differentiation state. How CBMs are able to bind to the backbone sugar polymer and yet display sufficient promiscuity in ligand recognition to accommodate these variable side chains is an important issue. Recent crystal structures of CBMs in complex with their target ligands have started to clarify how these proteins are able to bind to decorated ligands. Thus, the crystal structure of CBM15 in complex with xylopentaose showed that six of the ten C2-OH and C3-OH moieties were solvent exposed (Szabo et al., 2001), demonstrating how this protein was able to interact with xylose polymers that contained numerous side chains. Similarly, the structure of CBM29 bound to mannohexaose showed that the C6-OH groups of alternate mannose residues were solvent exposed, showing how this protein was able to interact with mannan that was decorated at C6 with galactosyl side chains (Charnock et al., 2002). The structures of CBM13 (Notenboom et al., 2002; Fujimoto et al., 2002) and CBM6 (Boraston et al., 2003a) bound to xylooligosaccharides also reveal information on the extent to which the side chains of xylans can be accommodated in the binding site of these proteins. The paper by Boraston et al. (2003b) presented in this issue of Structure makes a decisive contribution to understanding the mechanism by which CBMs are able to bind to decorated ligands. This group solved the structure of a novel family 27 mannan binding CBM in complex with mannohexaose and 63,64-␣-D-galactosylmannopentaose. The structure revealed how the protein is able to accommodate mannan, a 1,4-linked mannose polymer as opposed to cellulose, a 1,4-linked glucose polymer. The CBM makes numerous hydrogen bonds with the C2-OH of the mannose sugars, and predicted steric clashes between C2-OH equatorial sugar hydroxyls and the protein, which would preclude glucose from binding at subsites 3 and 4, provides an explanation for why this protein binds only to mannose-containing polymers. It is interesting to note that despite not binding to cellulooligosaccharides, or indeed accommodating glucose at two of the five subsites, the protein is able to bind the mixed glucose-mannose polymer glucomannan with essentially complete coverage. The Boraston paper importantly provides the first glimpses of a CBM bound to a decorated oligosaccharide. The galactosyl side chains can be accommodated in subsites 1, 2, and 5 where the C6-OH groups point into solvent. In subsite 4, the galactose side chains extend out from the edges of the ribbon formed by the 2-fold axis of the -1,4-linked mannose structure, consistent with the known structure of galactomannan. The galactose attached to the mannose in subsite 3, however, is located in a plane parallel to the mannose backbone. This galactose appears to be forced into this conformation by Trp-28, whose position would not allow the galactose side chain to extend away from the mannan backbone, and thus provides an explanation for the low
affinity toward the decorated ligand. These data demonstrate how CBM27 is able to bind a highly decorated galactomannan, as four of the five subsites are able to accommodate the sugar side chain. The Boraston paper also makes a significant contribution to our understanding of the stoichiometry of CBM ligand binding. The authors provide compelling biochemical evidence that at high protein concentration, two molecules of the CBM are bound to one molecule of mannohexaose. The oligosaccharide binds the two CBM molecules sequentially. The first binding event has a high affinity, whereas the second protein interacts only weakly with the CBM27-mannohexaose complex. The structural basis for this dimer-ligand complex is unclear, as no crystallographic data on this trimolecular complex are available. It is possible that the oligosaccharide spans the two CBMs and thus partially occupies the binding cleft of the two proteins. Such a binding event, however, would require a significant redistribution of the oligosaccharide that is not associated by a highly beneficial ⌬G. An alternative, more likely, hypothesis is that the second CBM molecule binds to the faces of the oligosaccharide that are not interacting with the already bound protein. Thus, even if this second CBM27-oligosaccharide interaction is weak, it does not compromise the initial high-affinity CBM27-mannohexaose binding event. Clearly, structural data are required to elucidate which mechanism is correct. The paper by Boraston et al. has made an important contribution to the mechanism by which CBMs are able to accommodate decorated polysaccharides, and provides new insights into the assembly of CBM-oligosaccharide complexes, and as such represents a landmark discovery in the field of carbohydrate protein recognition. Harry J. Gilbert School of Cell and Molecular Biosciences University of Newcastle upon Tyne Newcastle upon Tyne NE1 7RU United Kingdom Selected Reading Boraston, A.B., Nurizzo, D., Notenboom, V., Ducros, V., Rose, D.R., Kilburn, D.G., and Davies, G.J. (2002). J. Mol. Biol. 319, 1143–1156. Boraston, A.B., Notenboom, V., Warren, R.A., Kilburn, D.G., Rose, D.R., and Davies, G. (2003a). J. Mol. Biol. 327, 659–669. Boraston, A.B., Revett, T.J., Boraston, C.M., Nurizzo, D., and Davies, G.J. (2003b). Structure 11, this issue, 665–675. Charnock, S.J., Bolam, D.N., Nurizzo, D., Szabo, L., McKie, V.A., Gilbert, H.J., and Davies, G.J. (2002). Proc. Natl. Acad. Sci. USA 99, 14077–14082. Fujimoto, Z., Kuno, A., Kaneko, S., Kobayashi, H., Kusakabe, I., and Mizuno, H. (2002). J. Mol. Biol. 316, 65–78. Notenboom, V., Boraston, A.B., Chiu, P., Freelove, A.C., Kilburn, D.G., and Rose, D.R. (2001). J. Mol. Biol. 314, 797–806. Notenboom, V., Boraston, A.B., Williams, S.J., Kilburn, D.G., and Rose, D.R. (2002). Biochemistry 41, 4246–4254. Simpson, P.J., Xie, H., Bolam, D.N., Gilbert, H.J., and Williamson, M.P. (2000). J. Biol. Chem. 275, 41137–41142. Szabo, L., Jamal, S., Xie, H., Charnock, S.J., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2001). J. Biol. Chem. 276, 49061–49065. Tormo, J., Lamed, R., Chirino, A.J., Morag, E., Bayer, E.A., Shoham, Y., and Steitz, T.A. (1996). EMBO J. 15, 5739–5751.