Crystal structure of a glycoside hydrolase family enzyme, CcCel6C, a cellulase constitutively produced by Coprinopsis cinerea Yuan Liu1, Makoto Yoshida1, Yuma Kurakata2, Takatsugu Miyazaki2, Kiyohiko Igarashi3, Masahiro Samejima3, Kiyoharu Fukuda1, Atsushi Nishikawa2 and Takashi Tonozuka2 Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Japan Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Japan Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan Keywords basidiomycete; cellobiohydrolase; cellulase induction; endoglucanase; glycoside hydrolase family Correspondence T Tonozuka, Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Fax: +81 42 367 5705 Tel: +81 42 367 5702 E-mail: tonozuka@cc.tuat.ac.jp (Received 18 November 2009, revised January 2010, accepted 14 January 2010) doi:10.1111/j.1742-4658.2010.07582.x The basidiomycete Coprinopsis cinerea produces the glycoside hydrolase family enzyme CcCel6C at low and constitutive levels CcCel6C exhibits unusual cellobiohydrolase activity; it hydrolyses carboxymethyl cellulose, which is a poor substrate for typical cellobiohydrolases Here, we determined the crystal structures of CcCel6C unbound and in complex with p-nitrophenyl b-d-cellotrioside and cellobiose CcCel6C consists of a distorted seven-stranded b ⁄ a barrel and has an enclosed tunnel, which is observed in other cellobiohydrolases from ascomecetes Hypocrea jecorina (HjeCel6A) and Humicola insolens (HinCel6A) In HjeCel6A and HinCel6A, ligand binding produces a conformational change that narrows this tunnel In contrast, the tunnel remains wide in CcCel6C and the conformational change appears to be less favourable than in HjeCel6A and HinCel6A The ligand binding cleft for subsite )3 of CcCel6C is also wide and is rather similar to that of endoglucanase These results suggest that the open tunnel and the wide cleft are suitable for the hydrolysis of carboxymethyl cellulose Introduction Cellulose, a linear polymer made up of glucose units linked by b-1,4-glucosidic linkages, is the predominant structural component of plant cell walls and is the most abundant biomass resource on Earth Cellulases hydrolyse the b-1,4-glucosidic bonds of cellulose chains and are traditionally classified as endoglucanases (EC 3.2.1.4) or cellobiohydrolases (EC 3.2.1.91) based on their activity profiles Endoglucanases randomly cleave the internal b-1,4-glucosidic bond of cellulose, whereas cellobiohydrolases preferentially act on the end of the chain and progressively cleave off cellobiose as the main product [1–3] Cellulases belonging to the glycoside hydrolase family (GH6) are known as major cellulolytic enzymes produced by filamentous fungi For example, GH6 cellulases from the best studied cellulolytic organism, ascomycete Hypocrea jecorina (formerly known as Trichoderma reesei), make up 12–20% of total extracellular protein when the fungus grows in cellulolytic culture [4] Therefore, GH6 enzymes have been considered attractive enzymes for industrial application, such as biomass conversion The CAZy database (http://www.cazy.org/) [5] broadly categorizes the GH6 enzymes into cellobiohydrolasetype and endoglucanase-type enzymes In 1990, the first Abbreviations CcCel6C, Coprinopsis cinerea Cel6C; GH, glycoside hydrolase family; (Glc)2-S-(Glc)2, methylcellobiosyl-4-thio-b-cellobioside; HinCel6A, Humicola insolens Cel6A; HinCel6B, Humicola insolens Cel6B; HjeCel6A, Hypocrea jecorina Cel6A; PcCel7A, Phanerochaete chrysosporium, Cel7A; pNPG2, p-nitrophenyl b-D-cellobioside; pNPG3, p-nitrophenyl b-D-cellotrioside 1532 FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Liu et al crystal structure of a cellulase was reported; it was a catalytic domain of Hypocrea jecorina Cel6A (HjeCel6A, formerly designated cellobiohydrolase II), a GH6 cellobiohydrolase from an ascomycete [6] In that same decade, the crystal structure of the catalytic domain in HinCel6A, another GH6 cellobiohydrolase from ascomycete Humicola insolens, was determined [7] The catalytic domains of HjeCel6A and HinCel6A consist of a distorted seven-stranded b ⁄ a barrel; the striking feature is that they have active sites enclosed by N-terminal and C-terminal loops that form a tunnel The enclosed active sites trap the cellulose chain in the tunnel and delay enzyme–substrate dissociation, which promotes the cleavage of several sequential substrate bonds [8,9] In contrast and despite displaying higher sequence similarity to HjeCel6A and HinCel6A, the structure of a fungal endoglucanase, Humicola insolens Cel6B (HinCel6B), shows active sites in a cleft formed by a C-terminal loop deletion coupled with the peeling open of an N-terminal loop [10] Many reports are available on the crystal structures of the GH6 enzymes from ascomycetes, but no crystal structure of the basidiomycete-derived GH6 enzyme has yet been determined Recently, we cloned five genes encoding GH6 enzymes from a basidiomycete Coprinopsis cinerea (formerly known as Coprinus cinereus), and the enzymes have been designated CcCel6A, CcCel6B, CcCel6C, CcCel6D and CcCel6E [11] The amino acid sequences corresponding to the active site enclosing loops of cellobiohydrolases have been observed in all five enzymes In the evolutionary tree, however, four of the enzymes, CcCel6B–6E, have mapped to a region distant from CcCel6A There are high sequence identities of CcCel6A–HjeCel6A (48%) and CcCel6A–HinCel6A (52%), including an N-terminal cellulose binding domain In contrast, CcCel6B–6E fall into a region closer to the endoglucanase HinCel6B in the evolutionary tree, and no cellulose binding domain is found in the four enzymes For example, the sequence identities of CcCel6C–HjeCel6A and CcCel6C–HinCel6A are 36 and 39%, respectively, whereas that of CcCel6C–HinCel6B is 43% Transcript analysis showed that the presence of cellobiose strongly induced transcription of the CcCel6A gene, but weakly induced transcription of the CcCel6B, -6D and -6E genes Interestingly, the transcript level of CcCel6C was not influenced by either glucose or cellobiose When the enzymatic properties were investigated, CcCel6B and CcCel6C exhibited cellobiohydrolase activity, but the enzymes hydrolysed carboxymethyl cellulose, which is a poor substrate for typical GH6 cellobiohydrolases [12] These results indi- Structure of C cinerea CcCel6C cate that the physiological function and the substrate binding mechanism of CcCel6C are expected to be different from those of known cellobiohydrolases Here, we present the crystal structure of CcCel6C To our knowledge, this is the first report of the crystal structure of a basidiomycete GH6 enzyme Results and Discussion Overall structures of CcCel6C The crystal structures of unliganded CcCel6C and the enzyme–substrate complexes of CcCel6C–p-nitrophenyl b-d-cellotrioside (pNPG3) and CcCel6C–cellobiose ˚ were determined at 1.6, 1.4 and 1.2 A resolutions, respectively (Table 1) The crystal belongs to the space group P1, which contains one molecule in an asymmetric unit In Ramachandran plots, 95.7% (unliganded CcCel6C), 95.7% (CcCel6C–pNPG3) and 95.4% (CcCel6C–cellobiose) of residues were shown to be in favoured regions, and no residues were identified as outliers, as calculated by the molprobity server [13] The electron density (2Fo–Fc) maps contoured at 1r show continuous density for almost all main chain atoms except for the first  12 N-terminal residues and the last  12 C-terminal segments containing the His-tag sequence The overall structure of CcCel6C alone is shown in Fig 1A Like the fungal GH6 cellobiohydrolases [6,7], CcCel6C consists of a sevenstranded b ⁄ a barrel fold a-Helices and b-strands are numbered as a1–a8 and b0–bVII, respectively, as shown in Fig 2, based on the numbering scheme for HinCel6A [7] Structural homology was researched using the dali server [14] and CcCel6C was found to most resemble the fungal GH6 enzymes: HinCel6A (cellobiohydrolase; Z score, 54.2) [7], HjeCel6A (cellobiohydrolase; Z score 54.1) [6], HinCel6B (endoglucanase; Z score, 51.1) [10] and bacterial GH6 enzymes (e.g 1UOZ [15] and 1TML [16]; Z score  30) The Ca backbone of unliganded CcCel6C was superposed with those of cellobiohydrolases HjeCel6A, HinCel6A and endoglucanase HinCel6B using the program superpose in the ccp4 suite [17] The results indicated that the folds of CcCel6C are almost identical to not only the cellobiohydrolases, but also to the endoglucanase (Fig 1B) ˚ The rmsd values are 1.16 A (CcCel6C–HinCel6A, ˚ 1BVW), 1.14 A (CcCel6C–HjeCel6A, 1QK0 chain A) ˚ and 1.30 A (CcCel6C–HinCel6B, 1DYS chain A) for main chain atoms The significant feature in cellobiohydrolases HjeCel6A and HinCel6A is their active site located inside an enclosed tunnel [6–10,18]; CcCel6C has a FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS 1533 Structure of C cinerea CcCel6C Y Liu et al Table Data collection and refinement statistics Unliganded Data collection Beamline Space group Cell dimensions ˚ a (A) ˚ b (A) ˚ c (A) a (°) b (°) c (°) ˚ Resolution range (A) Measured reflections Unique reflections Completeness (%) I ⁄ r(I) Rmerge Refinement statistics Rwork Rfree rmsd ˚ Bond lengths (A) Bond angles (°) Number of atoms Protein Ligand Magnesium Water ˚ Average B (A2) Protein Ligand Magnesium Water a Cellobiose pNPG3 PF-AR NW12A P1 PF-AR NW12A P1 PF-AR NW12A P1 44.0 45.1 48.9 77.8 87.3 68.8 50–1.60 (1.66–1.60)a 85 577 43 370 95.8 (94.0)a 33.2 (9.2)a 0.025 (0.090)a 44.2 45.4 49.1 77.6 86.9 68.6 50–1.20 (1.24–1.20)a 197 765 100 613 92.9 (89.4)a 29.4 (3.7)a 0.036 (0.184)a 43.9 45.2 49.0 77.6 86.8 68.8 50–1.40 (1.45–1.40)a 125 859 63 990 94.8 (90.6)a 25.7 (3.3)a 0.064 (0.175)a 0.141 0.165 0.148 0.169 0.163 0.189 0.008 1.12 0.008 1.20 0.008 1.22 2894 – 469 2959 35 579 2940 64 494 15.7 – 32.7 30.1 12.9 23.9 18.1 25.7 12.5 33.1 15.3 25.8 The values for the highest resolution shells are given in parentheses homologous tunnel and its conformation is very similar in the liganded and unliganded enzymes (Fig 1C) Two loops, loop-1 and loop-2, forming this tunnel are identified between bII and a4 and between bVII and a8’ (Fig 2) Loop-1 and loop-2 contain disulfide bridges of Cys103–Cys164 and Cys298– Cys348, respectively, like those seen in HjeCel6A and HinCel6A Although the entire backbones of CcCel6C, HjeCel6A and HinCel6A are essentially identical, the two loops of the three enzymes are not exactly superposed (Fig 1B) In HinCel6A [7], a magnesium ion forms a hexa-coordinated geometry involved in the crystal contacts and the same magnesium-mediated geometry is found in the unliganded CcCel6C, CcCel6C–pNPG3 and CcCel6C–cellobiose Here, this magnesium ion is located close to Asp109 in loop-1 Another hexa-coordinated magnesium ion, which is observed near Glu33, is found only in CcCel6C–cellobiose 1534 Ligand-bound structures Comparing the ligand-bound structures of HjeCel6A and HinCel6A with CcCel6C–pNPG3 and CcCel6C– cellobiose enabled us to label the subsites of CcCel6C In CcCel6C–pNPG3, electron density for two ligand molecules was seen in the active site (Fig 3A) The molecule bound to subsites )3 to )1 was modelled as p-nitrophenyl b-d-cellobioside (pNPG2, not pNPG3) ˚ (average B = 25.4 A2; Figs 3A, 4A), and a glucose unit at the nonreducing end of pNPG3 was not identified in the difference Fourier map The other molecule bound to subsites +2 to +4 was also modelled as pNPG2 (Figs 3A, 4B), but the map is better resolved ˚ at the lower contoured level (average B = 40.8 A2) Weak electron density is present at subsite +1, but we could not place the models In CcCel6C–cellobiose, electron density for two ligand molecules, a cellobiose molecule bound to subsites +1 and +2 and a glucose FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Liu et al Structure of C cinerea CcCel6C A R343 D109 D150 R343 D109 D150 B Fig Overall structures of CcCel6C (A) Stereoview of CcCel6C–cellobiose shown as a ribbon model a-Helices, b-strands and disulfide bridges are indicated in blue, orange and green, respectively Cellobiose and glucose molecules bound to the active site are shown in red Side chains of Asp109, Asp150 and Arg343 are shown in pink (B) Stereoview of the Ca backbone of CcCel6C–cellobiose (red), which is superposed on those of HjeCel6A–(Glc)2-S-(Glc)2 (yellow; PDB id, 1QK2), HinCel6A–cellobiose (cyan; PDB id, 2BVW) and the endoglucanase HinCel6B (gray; PDB id, 1DYS) The ligands bound to the active site are illustrated as stick models (C) Comparison of the Ca backbones of unliganded CcCel6C (blue), CcCel6C–pNPG3 (green) and CcCel6C–cellobiose (red) C molecule bound to subsite )2, were identified (Fig 3B) The cellobiose molecule gives a clear elec˚ tron density map (average B = 23.0 A2), whereas part of the density for the glucose molecule is not visible ˚ (average B = 25.8 A2) A similar single glucose molecule has been found in subsite )2 of the HinCel6A– cellobiose complex, but it remains unclear whether the glucose molecule is part of cellobiose or from contamination in the commercial cellobiose preparation [18] The structures of CcCel6C–pNPG3, CcCel6C–cellobiose, HinCel6A–cellobiose [18] and HjeCel6A–methylcellobiosyl-4-thio-b-cellobioside [(Glc)2-S-(Glc)2] [8] were superposed to depict the characteristics of the ligand binding site of CcCel6C (Fig 3C) The glucose units in subsites )2, +1, +2 and +3 (abbreviated as Glc )2, +1, +2 and +3, respectively) overlaid well, whereas the aromatic ring of pNPG3 in subsite )1 was at a position markedly different from that of Glc )1 Study of the HjeCel6A–(Glc)2-S-(Glc)2 complex has shown that Glc )1 adopts a distorted conformation, and many hydrogen bonds between HjeCel6A and Glc )1 appear to stabilize this energetically unfavoured conformation [8] The p-nitrophenyl group of pNPG3, however, is not able to form similar hydrogen bonds with CcCel6C, resulting in the different position in the active site Although controversy exists concerning the active site residues of GH6 enzymes [19], two Asp residues are suggested to be catalytic [7] The sequence alignment of HjeCel6A, HinCel6A and CcCel6C (Fig 2) indicated that Asp150 and Asp334 of CcCel6C are the potential catalytic residues and could act as a proton donor and a base, respectively Another aspartic acid residue (Asp175 of HjeCel6A) has been proposed to contribute to the electrostatic stabilization of the partial positive charge in the transition state FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS 1535 Structure of C cinerea CcCel6C Y Liu et al Fig Comparison of amino acid sequences of CcCel6C and related enzymes The sequences were aligned using the CLUSTALW2 server, and manual adjustment was carried out based on the comparison of the crystal structures The numbering of amino acid residues and secondary structures (a1–a8 and b0–bVII) are given Residues listed in Table are printed with a red ⁄ pink (subsites )3 to )1) or blue ⁄ cyan (subsites +1 to +4) background The two loops, loop-1 and loop-2, are underlined Other symbols: arrow, Asp150 and Arg343; asterisk, three conserved aspartic acid and four conserved tryptophan described in the text; dashed line, disulfide bridge [20], and a homologous residue in CcCel6C is probably Asp102 Two distinct conformations for the catalytic acid have been observed in HjeCel6A and HinCel6A, and both are proposed to be important in the catalysis [7,20] The Fo–Fc omit map shows that these two conformations are present for Asp150 in CcCel6C–cellobiose (Fig S1A), but in CcCel6C– pNPG3, one of the conformations is not seen, probably due to steric hindrance with the p-nitrophenyl group of pNPG3 To probe the interaction between CcCel6C and the ligands, CcCel6C–pNPG3 and CcCel6C–cellobiose were analysed using the program ligplot [21], and taken together, 21 amino acid residues appear to participate in ligand binding Table lists these residues, plus the three conserved aspartic acid residues proposed to be involved in catalysis The amino acid residues in subsites )2 to +4 are highly conserved among CcCel6C, HjeCel6A, HinCel6A and HinCel6B Four key tryptophan residues involved in substrate stacking interactions are fully conserved in CcCel6C as Trp61 (subsite )2), Trp297 (+1), Trp198 (+2) and Trp201 (+4) (Fig 2) as previously described [9,22] A tyrosine 1536 residue critical for the distortion of Glc )1 (Tyr169 in HjeCel6A) [23–25] is identified in CcCel6C as Tyr86 The enclosed tunnel The cellobiohydrolases have been characterized by the enclosed tunnel as described above A conspicuous feature of CcCel6C is that the enclosed tunnel is wider than those of HjeCel6A and HinCel6A (Fig 5A–C), and loop-1 and loop-2 of CcCel6C revealed a more open structure than those of HjeCel6A and HinCel6A The conformational changes in the two loops of HjeCel6A and HinCel6A have been reported; binding of the ligands such as (Glc)2-S-(Glc)2 or cellobiose results in a narrowing of the tunnel, and the additional empty space is not seen in the vicinity of the ligands [8,18] In CcCel6C, however, the two loops of the unliganded CcCel6C, CcCel6C–pNPG3 and CcCel6C–cellobiose are superposed well (Fig 1C), and the rmsd values for ˚ all backbone atoms are 0.109 A (between unliganded ˚ CcCel6C and CcCel6C–pNPG3) and 0.159 A (between unliganded CcCel6C and CcCel6C–cellobiose) The possibility that the two loops are trapped in the open FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Liu et al A –3 Structure of C cinerea CcCel6C –2 –1 +1 +2 +3 +4 A B C K30 D334 S26 W61 S236 W201 W297 D150 Y96 W198 D102 B Fig Comparison of the ligands bound to the active site (A) The pNPG3 Fo–Fc electron density maps at the 2.0 r contoured level The subsite numbers are labelled from )3 to +4 (B) The cellobiose Fo–Fc electron density maps at the 2.0 r contoured level (C) Overlays of the ligands in CcCel6C–pNPG3 (green), CcCel6C–cellobiose (red), HjeCel6A–(Glc)2-S-(Glc)2 (yellow; PDB id, 1QJW) and HinCel6A–cellobiose (cyan; PDB id, 2BVW) Some critical residues described in the text are indicated in black conformations by crystal packing could not be excluded, as the crystals of the complex structures were obtained by soaking with pNPG3 or cellobiose In HjeCel6A–(Glc)2-S-(Glc)2, however, the conformational changes in the tunnel-forming loops have been observed by addition of the ligand after the crystals had reached full size [8] The most significant movement of HinCel6A occurs at residues Ala183 to Gly188 [18], and the sequence of HjeCel6A ⁄ HinCel6A, A-L ⁄ A-A-S-N-G, is composed of amino acids with relatively small side chains The corresponding region of CcCel6C (residues 105–110) is A-K-A-S-D-G, which contains a bulky lysine residue It appears that the conformational change in the two loops of CcCel6C is less favourable and the tunnel still has an open space near the binding sites of cellobiose or pNPG3 Loop-1 contacts with loop-2 mainly via an interaction between Asp109 and Arg343 In unliganded CcCel6C, the electron density map for Asp109 is clear, but the 2Fo–Fc map for Arg343 is better resolved at the lower contoured level of 0.8r, and two hydrogen bonds, Asp109 OD2-Arg343 NE and Ser108 O-Arg343 NH1 could form directly between loop-1 and loop-2 in this model As for Asp109 in both CcCel6C–pNPG3 and CcCel6C–cellobiose and Arg343 in CcCel6C– Fig Schematic drawing of the amino acid residues interacting with the ligands observed at subsites )3 to )1 (A) and +2 to +4 (B) Symbols: open circle, oxygen atom; closed circle, carbon atom; gray circle, nitrogen atom; dashed line, hydrogen bond The residues involved in hydrophobic interactions are illustrated pNPG3, the Fo–Fc omit maps show that there are at least two different conformations (Fig S1B) This observation suggests that the enclosed tunnel of CcCel6C is not completely ‘enclosed’, although the ligand molecules are unable to pass through the opening between loop-1 and loop-2 From subsites )2 to +4, only one serine residue, Ser236, is not conserved in the other fungal cellobiohydrolases (Table 2), and the corresponding residue of HjeCel6A and HinCel6A is alanine (Ala304 and Ala309, respectively) As described in the previous section, the tunnel-forming loops of HinCel6A are changed to adopt the closed conformation when the ligands bind to the active site As a result, in HinCel6A–cellobiose, a serine residue in loop-1, FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS 1537 Structure of C cinerea CcCel6C Y Liu et al Table Amino acid residues interacting with the ligands or potentially involved in the catalysis, and the corresponding residues of HjeCel6A, HinCel6A and HinCel6B pNP, p-nitrophenyl group Closest Glc ⁄ pNPa Glc )3 Glc )2 pNP )1 Glc +1 Glc +2 Glc +3 pNP +4 CcCel6C HjeCel6A HinCel6A HinCel6B S26 K30 E332 G361 W61 R101 K328 P329 Y96 S236 D102 D334 D150 N237 W297 N158 H195 G197 W198 W294 G295 T157 N204 W201 (Y103)b (E107)b E399 G428 W135 R174 K395 P396 Y169 (A304)b D175 D401 D221 N305 W367 N229 H266 G268 W269 W364 G365 T228 N257 W272 (Y104)b (E108)b E403 G432 W137 R179 K399 P400 Y174 (A309)b D180 D405 D226 N310 W371 N234 H271 G273 W274 W368 G369 T233 N280 W277 (D16)b K20 E314 G328 W52 R91 K310 P311 Y86 S221 D92 D316 D139 N222 W282 (G147)b (N183)b G185 W186 W279 G280 T146 (K192)b W189 a The closest Glc ⁄ pNP is determined based on the cartoon generated using the program LIGPLOT b Amino acid residues that are not identical to those of CcCel6C are given in parentheses Ser186, can directly form hydrogen bonds with the ligand [18] The conformational change of HjeCel6A– (Glc)2-S-(Glc)2 has been reported to be more compliA D 1538 B E cated, and four states (most closed, more open, even more open, and most open) of the loop have been identified The complex of wild-type HjeCel6A–(Glc)2S-(Glc)2 (PDB id, 1QK2) has been observed in the ‘more open’ form and the corresponding serine residue, Ser181, does not interact with (Glc)2-S-(Glc)2 The Y169F mutant of HjeCel6A complexed with (Glc)2-S(Glc)2 (PDB id, 1QJW), on the other hand, adopts the ‘most closed’ form, and Ser181 is pointed into the )1 site and OG atom of the serine residue hydrogen bonds with O5 of Glc )1, O4 of Glc )1, and O2 of Glc )2 [8] It is not easy to interpret the role of the Ser181 ⁄ 186 residue during the catalysis, but they appear to stabilize the distorted conformation of Glc )1 However, the significant conformational changes of the two loops of CcCel6C were not observed (Fig 1C) and in CcCel6C–cellobiose, Ser108, the position equivalent to Ser181 ⁄ Ser186, does not directly hydrogen bond with the ligand In the endoglucanases, the corresponding residue of Ser236 is found to be serine (Ser221, HinCel6B; Ser189, Thermobifida fusca Cel6A) and in the complex of Thermobifida fusca, Cel6A with (Glc)2-S-(Glc)2, Ser189 hydrogen bonds with O6 of the distorted glucose unit Glc )1 [25] The role of Ser236 in CcCel6C is probably similar to that of Ser189 in Thermobifida fusca Cel6A Subsite )3 In contrast to the high similarity of subsites from )2 to +4, two amino acid residues involved in subsite )3 of CcCel6C, Ser26 and Lys30, are strikingly different from those of HjeCel6A (Tyr103 and Glu107, respectively) and HinCel6A (Tyr104 and Glu108, C F Fig Surface models of CcCel6C and related enzymes (A–C) Overall structures of CcCel6C (A), HinCel6A (B) and the endoglucanase HinCel6B (C) (D–F) Close-up views in the vicinity of subsite )3 of CcCel6C (D), HinCel6A (E) and HinCel6B (F) To generate the models, the structure of CcCel6C–pNPG3 was superposed on those of HinCel6A (PDB id, 2BVW) and HinCel6B (PDB id, 1DYS), and pNPG2 was placed on the models Glc )3 is labeled as )3 Residues forming protruding knobs at the entrance of the cleft are shown in yellow and red, and the distances between them ˚ (A) are indicated Ser26 and Lys30 of CcCel6C and the corresponding residues are indicated in cyan and pink, respectively FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Liu et al respectively) For the cellobiohydrolases, subsite )3 is typically presumed unnecessary to produce cellobiose, but the studies of HinCel6A have revealed no substantial evidence to completely negate a )3 subsite [18] and subsites from )4 to +4 of HinCel6A are proposed [9] In CcCel6C, Glc )3 of pNPG3 not only forms multiple hydrogen bonds with Ser26, Lys30 and Trp61 through water molecules, but also makes hydrophobic contacts with Pro329, Glu332 and Gly361 (Fig 5D) However, modelling pNPG3 in its CcCel6C-bound conformation with either HjeCel6A or HinCel6A, Glc )3 causes steric conflict with a tyrosine residue (HjeCel6A Tyr103 and HinCel6A Tyr104) (Fig 5E) The cleft for subsite )3 of CcCel6C is also apparently wider than those of HjeCel6A or HinCel6A (Fig 5A, B) Asp64 and Glu362 of CcCel6C form protruding knobs at the entrance of the cleft, and the distance between atom OD2 of Asp64 and atom OE2 of ˚ Glu362 is 9.8 A (Fig 5D) Similar knobs, which are formed by Arg140 and Gln433, are observed at the entrance of the cleft of HinCel6A, but the distance between atom NH1 of Arg140 and atom NE2 of ˚ Gln433 is only 6.9 A (Fig 5E) These observations indicate that the accessibility of subsite )3 of CcCel6C is less restricted than those of HjeCel6A and HinCel6A The cleft for subsite )3 of CcCel6C is rather similar to that of the endoglucanase HinCel6B (Fig 5C, F) The residue of HinCel6B equivalent to Ser26 of CcCel6C is identified as Asp16, an amino acid residue with a relatively small side chain and, thus, no steric conflict is found if the similar placement is tested for the endoglucanase HinCel6B The width of the entrance of the cleft for subsite )3 (ND2 of Asn55-SD ˚ of Met329) is 11.6 A, which is similar to that of CcCel6C (Fig 5F) Implications for enzymatic activity The structure of CcCel6C contains the enclosed tunnel around its active site, indicating that the enzyme has cellobiohydrolase activity Indeed, our previous study showed that CcCel6C hydrolysed phosphoric acidswollen cellulose with the release of cellobiose as a main product [12] However, the enzyme lacks a cellulose binding domain, which is necessary to hydrolyse crystalline cellulose, and most GH6 cellobiohydrolases have this domain In addition, we have reported that the transcript level of CcCel6C was very low at the active growth stage in the cellulose-degrading culture, and almost the same transcript level was detected at the active growth stage in the glucose culture The transcript level also did not change when the mycelia were transferred to a medium containing glucose, Structure of C cinerea CcCel6C cellobiose or no carbon source [11] These findings suggest that the physiological role of CcCel6C does not involve the degradation of crystalline cellulose Cellulose is insoluble in water; for the enzyme to recognize it, the insoluble cellulose must be converted into soluble saccharides, such as cellobiose and cellooligosaccharides In the past several decades, it has been assumed that low and constitutive levels of cellulases react with cellulose to produce a soluble molecule that enters the cell and induces transcription of cellulase genes [26,27] Considering the results of our biochemical and transcript analyses, CcCel6C probably produces a small amount of cellobiose when cellulose is present Similar activity has been reported in a GH7 enzyme produced by basidiomycete Phanerochaete chrysosporium, PcCel7A [28–30] This enzyme shows the amino acid sequence corresponding to an active site tunnel also shown in GH7 cellobiohydrolases, but like CcCel6C, lacks a cellulose binding domain A low level of PcCel7A transcripts was observed in the culture containing cellulose, whereas the transcripts were detected in glucose culture In a homology modelling analysis, the enzyme was expected to have endo-type activity [31] These enzymatic and transcriptional properties are very similar to those of CcCel6C and, thus, PcCel7A might also produce an inducer of cellulase genes Recently, it was also reported that basidiomycete Coniophora puteana has GH6 and GH7 enzymes without a cellulose binding domain [32] Therefore, the existence of cellobiohydrolases lacking a cellulose binding domain might characterize soluble cellulose degradation specifically in basidiomycetes because this type of enzyme was not found in ascomycetes Many reports have shown that cellobiohydrolases are typically active on crystalline cellulose [1] The fungal cellobiohydrolases, HjeCel6A and HinCel6A, have the cellulose binding domain [33,34] Specific conformational changes have been observed in the two active site enclosing loops of HjeCel6A and HinCel6A that seem to be critical in hydrolysing the crystalline cellulose [8,18] CcCel6A, whose amino acid sequence is highly similar to those of HjeCel6A and HinCel6A, probably has similar activity on amorphous cellulose in Coprinopsis cinerea The structures determined here indicate that CcCel6C has an enclosed tunnel similar to that of HjeCel6A and HinCel6A The tunnel is, however, wider and more open than these fungal cellobiohydrolases, and virtually no conformational change in the two loops of CcCel6C is induced The ligand binding cleft of CcCel6C is also wider due to the absence of the bulky tyrosine residue in subsite )3 (Fig 5), and the structures of subsites )1 and )3 of CcCel6C resem- FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS 1539 Structure of C cinerea CcCel6C Y Liu et al ble those of the endoglucanases HinCel6B rather than HjeCel6A and HinCel6A We have reported that CcCel6C hydrolyses the chemically modified cellulose derivative, carboxymethyl cellulose, whereas CcCel6A does not [12] The open tunnel and the wide cleft are probably suitable for the hydrolysis of carboxymethyl cellulose Carboxymethyl cellulose and amorphous cellulose have been reported to be substrates of most endoglucanases, indicating that the enzyme activity is mainly directed towards amorphous regions in the cellulose molecule [1] The results described above lead us to conclude that the architecture of CcCel6C could be suitable for hydrolysing amorphous cellulose to serve cellobiose, an inducer for the expression of CcCel6A in Coprinopsis cinerea [38], and the models for the ligands were built from both 2Fo–Fc and Fo–Fc electron density maps Solvent molecules were introduced using the program arp ⁄ warp Refinement statistics are listed in Table Superpositioning of CcCel6C with other protein structures and calculation of the rmsd values were carried out using the program superpose in the ccp4 suite Sequence identities were calculated using the program clustalw2 on the ebi server (http://www.ebi.ac.uk/Tools/clustalw2/) [39] with the default values Figures were generated using ligplot [21] and pymol (http:// www.pymol.org/) The coordinates and structure factors of unliganded CcCel6C, CcCel6C–pNPG3 and CcCel6C–cellobiose have been deposited in the Protein Data Bank under the accession codes 3A64, 3ABX and 3A9B, respectively Acknowledgements Experimental procedures Enzyme preparation and crystallization The expression and purification of CcCel6C were carried out as described previously [35] Briefly, recombinant CcCel6C fused with a His-tag was produced in Escherichia coli BL21(DE3) cells and purified with Ni-NTA agarose (Qiagen, Hilden, Germany) The enzyme was crystallized at 20 °C using the hanging drop vapour diffusion method, where lL CcCel6C (21.5 mgỈmL)1) was mixed with the same volume of well solution (100 mm Hepes ⁄ KOH pH 7.0, 30% polyethylene glycol 8000, 150 mm magnesium acetate) The obtained crystal was transferred to a cryo-solution of 40% (w ⁄ v) polyethylene glycol 8000 in well solution and flash frozen in a stream of nitrogen gas The crystal of the complex of pNPG3 (Seikagaku Corporation, Tokyo, Japan) or cellobiose was obtained by soaking in the same well solution (100 mm Hepes ⁄ KOH pH 7.0, 30% polyethylene glycol 8000, 150 mm magnesium acetate) containing 60 mm pNPG3 for h or 220 mm cellobiose for min, and the solution containing the ligand also acted as a cryoprotectant The diffraction data were collected at beamline PF-AR NW12 (Photon Factory, Tsukuba, Japan) The data were processed and scaled with the program hkl2000 [36] (Table 1) Model building and structure refinement The structure of CcCel6C was solved by molecular replacement with the program molrep in the ccp4 suite [17], and a model of HinCel6A (PDB id, 1BVW) [7] was employed as a probe model The automated model building was performed with the program arp ⁄ warp [37] The refinement 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should be addressed to the authors FEBS Journal 277 (2010) 1532–1542 ª 2010 The Authors Journal compilation ª 2010 FEBS ... et al crystal structure of a cellulase was reported; it was a catalytic domain of Hypocrea jecorina Cel 6A (HjeCel 6A, formerly designated cellobiohydrolase II), a GH6 cellobiohydrolase from an ascomycete... expression, crystallization and preliminary X-ray characterization of CcCel6C, a glycoside hydrolase family enzyme from the basidiomycete Coprinopsis cinerea Acta Crystallogr Sect F 65 , 140–143 36 Otwinowski... HinCel 6A and CcCel6C (Fig 2) indicated that Asp150 and Asp334 of CcCel6C are the potential catalytic residues and could act as a proton donor and a base, respectively Another aspartic acid residue