The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum  Pedro M Alzari, Hélè ne Souchon, Roberto Dominguez  Structure  Volume 4, Issue 3, Pages 265-275 (March 1996) DOI: 10.1016/S0969-2126(96)00031-7

Figure 1 Electron-density map of the catalytic center of uncomplexed CelA showing the environment of the two putative catalytic carboxylates. The map, contoured at 1.1σ, was calculated with observed amplitudes and DM-modified MIR phases at 1.9 å resolution. The refined model of CelA is superimposed. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 2 Overall view of the (α/α)6 barrel of endoglucanase CelA. (a) Side view of CelA showing the active-site cleft at the N-terminal end of the inner helices. The 12 α helices forming the barrel involve residues Gln52–Arg70, Ser94–Cys106, Gln110–Lys121, Thr151–Trp168, Tyr176–Cys191, Pro218–Thr228, Arg232–Val247, Tyr282–Phe293, Gln296–Ala310, Ala334–Ala343, Leu350–Ala362 and Tyr372–Ile384 (as defined by PROCHECK [35]). (b) Stereo Cα trace of CelA, viewed along the barrel axis. Amino acid positions are labeled every 20 residues. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 2 Overall view of the (α/α)6 barrel of endoglucanase CelA. (a) Side view of CelA showing the active-site cleft at the N-terminal end of the inner helices. The 12 α helices forming the barrel involve residues Gln52–Arg70, Ser94–Cys106, Gln110–Lys121, Thr151–Trp168, Tyr176–Cys191, Pro218–Thr228, Arg232–Val247, Tyr282–Phe293, Gln296–Ala310, Ala334–Ala343, Leu350–Ala362 and Tyr372–Ile384 (as defined by PROCHECK [35]). (b) Stereo Cα trace of CelA, viewed along the barrel axis. Amino acid positions are labeled every 20 residues. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 3 Stereoview of substrate binding to CelA. The difference maps, contoured at 3σ, were calculated at 1.9 å resolution with observed amplitudes (Fobsprotein+ligand−Fobsprotein) and DM-modified MIR phases. The refined model of bound ligand is shown in thick lines. The side chains of aromatic and acidic residues close to the sugar rings are shown as thin lines. (a) Complex of CelA with cellobiose (other cello-oligosaccharides produce similar difference maps). (b) Complex of CelA with IBTC. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 3 Stereoview of substrate binding to CelA. The difference maps, contoured at 3σ, were calculated at 1.9 å resolution with observed amplitudes (Fobsprotein+ligand−Fobsprotein) and DM-modified MIR phases. The refined model of bound ligand is shown in thick lines. The side chains of aromatic and acidic residues close to the sugar rings are shown as thin lines. (a) Complex of CelA with cellobiose (other cello-oligosaccharides produce similar difference maps). (b) Complex of CelA with IBTC. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 4 Protein–carbohydrate interactions in the CelA–cellobiose complex. (a) Stereoview showing stacking interactions between sugar rings and aromatic amino acid side chains. (b) Schematic diagram of atomic contacts. Hydrogen bonds are indicated with dashed lines, the corresponding distances are given in å. Several water molecules (labeled ‘Ow’) mediate enzyme-substrate interactions. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 4 Protein–carbohydrate interactions in the CelA–cellobiose complex. (a) Stereoview showing stacking interactions between sugar rings and aromatic amino acid side chains. (b) Schematic diagram of atomic contacts. Hydrogen bonds are indicated with dashed lines, the corresponding distances are given in å. Several water molecules (labeled ‘Ow’) mediate enzyme-substrate interactions. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 5 Substrate conformation. (a) Alternative orientation of adjacent β-1,4-linked glucosyl residues promotes the formation of a linear chain in cellulose. (b) Substrate binding to CelA imposes a similar orientation to consecutive glucopyranoside rings at subsites C and D, inducing a bend of the cellulose chain when within the active-site cleft. In the conformation shown, both the anomeric carbon C1 and the glycosidic oxygen O4 (indicated with arrows) can be approached from the same side of the substrate. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 5 Substrate conformation. (a) Alternative orientation of adjacent β-1,4-linked glucosyl residues promotes the formation of a linear chain in cellulose. (b) Substrate binding to CelA imposes a similar orientation to consecutive glucopyranoside rings at subsites C and D, inducing a bend of the cellulose chain when within the active-site cleft. In the conformation shown, both the anomeric carbon C1 and the glycosidic oxygen O4 (indicated with arrows) can be approached from the same side of the substrate. Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 6 View of the catalytic center of CelA showing the relative disposition of three carboxylate groups (Glu95, Asp152, and Asp278) close to the scissile glycosidic linkage and the two glucosyl residues bound at subsites B and D. Hydrogen bonds are represented by thin dashed lines. A possible orientation of the glucosyl ring at subsite C is shown in thick dashed lines. (The figure was drawn with MOLSCRIPT [38].) Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 7 Structural comparison of (α/α)6 glycosyl hydrolases. (a) Family 8 endoglucanase CelA. (b) Family 9 endoglucanase CelD from Clostridium thermocellum. (c) Family 15 glucoamylase-l from Aspergillus awamori. The figure shows a schematic view with α helices represented as cylinders (top) and the molecular surface of the active-site clefts colored according to charge (bottom). The coordinates of endoglucanase CelD (code 1 CLC) and glucoamylase-l (code 1 GLY) were taken from the Brookhaven Protein Data Bank [37]. (The figure was drawn with programs QUANTA [Molecular Simulations, Inc.] and GRASP [39].) Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 7 Structural comparison of (α/α)6 glycosyl hydrolases. (a) Family 8 endoglucanase CelA. (b) Family 9 endoglucanase CelD from Clostridium thermocellum. (c) Family 15 glucoamylase-l from Aspergillus awamori. The figure shows a schematic view with α helices represented as cylinders (top) and the molecular surface of the active-site clefts colored according to charge (bottom). The coordinates of endoglucanase CelD (code 1 CLC) and glucoamylase-l (code 1 GLY) were taken from the Brookhaven Protein Data Bank [37]. (The figure was drawn with programs QUANTA [Molecular Simulations, Inc.] and GRASP [39].) Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 7 Structural comparison of (α/α)6 glycosyl hydrolases. (a) Family 8 endoglucanase CelA. (b) Family 9 endoglucanase CelD from Clostridium thermocellum. (c) Family 15 glucoamylase-l from Aspergillus awamori. The figure shows a schematic view with α helices represented as cylinders (top) and the molecular surface of the active-site clefts colored according to charge (bottom). The coordinates of endoglucanase CelD (code 1 CLC) and glucoamylase-l (code 1 GLY) were taken from the Brookhaven Protein Data Bank [37]. (The figure was drawn with programs QUANTA [Molecular Simulations, Inc.] and GRASP [39].) Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)

Figure 8 Ramachandran plot of CelA. All main-chain dihedral angles occur in energetically allowed regions. Glycine residues are represented as filled triangles. (Produced with the program PROCHECK [35].) Structure 1996 4, 265-275DOI: (10.1016/S0969-2126(96)00031-7)