FEBS Letters 587 (2013) 2193–2198

journal homepage: www.FEBSLetters .org

Crystal structure of the N-terminal domain of a glycoside hydrolasefamily 131 protein from Coprinopsis cinerea

0014-5793/$36.00 � 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.febslet.2013.05.041

⇑ Corresponding author. Address: Department of Applied Biological Science,Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo183-8509, Japan. Fax: +81 42 367 5705.

E-mail address: tonozuka@cc.tuat.ac.jp (T. Tonozuka).

Takatsugu Miyazaki a, Makoto Yoshida b, Mizuki Tamura a, Yutaro Tanaka a, Kiwamu Umezawa b,Atsushi Nishikawa a, Takashi Tonozuka a,⇑a Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japanb Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 April 2013Revised 8 May 2013Accepted 9 May 2013Available online 24 May 2013

Edited by Miguel De la Rosa

Keywords:Glycoside hydrolase family 131Basidiomyceteb-GlucanaseCoprinopsis cinerea

The crystal structure of the N-terminal putative catalytic domain of a glycoside hydrolase family 131protein from Coprinopsis cinerea (CcGH131A) was determined. The structure of CcGH131A was foundto be composed of a b-jelly roll fold and mainly consisted of two b-sheets, sheet-A and sheet-B. Aconcave of sheet-B, the possible active site, was wide and shallow, and three glycerol molecules werepresent in the concave. Arg96, Glu98, Glu138, and His218 are likely to be catalytically critical resi-dues, and it was suggested that the catalytic mechanism of CcGH131A is different from that of typ-ical glycosidases.� 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Various enzymes have been reported to be involved in plantbiomass degradation. In 2012, an enzyme (Podospora anserinaglycoside hydrolase family 131 enzyme (PaGluc131A)) that exhib-its bifunctional exo-b-1,3-/-1,6- and endo-b-1,4 activities towardb-glucans was reported from an ascomycete fungus P. anserinaand was classified into the glycoside hydrolase (GH) family 131[1]. PaGluc131A is an unusual hydrolase, because the enzyme actson a broad range of b-glucan polysaccharides, including laminarin,curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives.Amino acid sequencing of PaGluc131A indicated that the enzymeconsists of a catalytic domain in the N-terminal part (residues1–252) and a carbohydrate-binding module family 1 (CBM1) do-main in the C-terminal part (307–345), and the two parts arejoined by a linker (253–306).

In the present study, we focused on a GH131 protein from abasidiomycete fungus Coprinopsis cinerea. The entire genomicsequence of C. cinerea has been reported [2]. The fungal genomecontains many genes coding for enzymes that participate in plant

cell biomass degradation, and we have previously reported thecrystal structures of cellobiohydrolases [3–5]. C. cinerea possessesa gene, CC1G_07166, which encodes a protein homologous toPaGluc131A (42% identity). We designated this protein as C. cinereaglycoside hydrolase family 131 protein (CcGH131A). The align-ment between the two proteins suggests that CcGH131A alsoconsists of a catalytic domain (residues 1–252) and a CBM domain(290–325), joined by a linker (253–289). In the present study, wedetermined the crystal structure of the N-terminal putative cata-lytic domain of CcGH131A, which represents the first crystal struc-ture of a GH131 protein.

2. Materials and methods

2.1. Molecular cloning

Total RNA was extracted from the C. cinerea strain 5338 [6], andfirst-strand cDNA was synthesized from total RNA as describedpreviously [7]. The N-terminal signal peptide (1–18) was predictedusing the SignalP server [8] (Center for Biological Sequence Analy-sis, Technical University of Denmark, http://www.cbs.dtu.dk/ser-vices/SignalP/). The DNA fragment encoding the GH131 domainwas amplified by PCR using the first-strand cDNA as a templateand the primers, 50-TTC ATA TGG GCC GTA TCG TGT GGG ATG-30

and 50-TTG CGG CCG CTC CAT CCC CAA TTT GGG TGG TAA TG-30

Table 1Data collection and refinement statistics.

Crystal form 1 Crystal form 2

Data collectionBeamline PF AR-NW12A PF AR-NW12AWavelength (Å) 0.97900 0.97905Space group P21 P1Cell dimensions

a (Å) 44.4 59.2b (Å) 68.2 68.4c (Å) 70.0 69.2a (�) 90.0 90.0b (�) 91.9 72.9c (�) 90.0 85.8

Resolution range (Å) 50–2.39 (2.48–2.39)a 50–2.0 (2.07–2.00)a

Unique reflections 16708 66894Redundancy 7.2 (6.2)a 4.2 (4.0)a

Completeness (%) 99.7 (97.9)a 94.7 (88.2)a

I/r(I) 40.0 (11.4)a 26.8 (7.6)a

Rmerge 0.125 (0.269)a 0.105 (0.289)a

Refinement statisticsRwork 0.188Rfree 0.236

RMSDBond lengths (Å) 0.009Bond angles (�) 1.270

Number of atomsProtein 7575Glycerol 72Water 787

Average B (Å2)Protein 15.6Glycerol 12.4Water 21.1

Ramachandran plot (Molprobity)Favored (%) 96.4Outliers (%) 0

RMSD, root-mean-square deviation.a The values for the highest resolution shells are given in parentheses.

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(the restriction sites of NdeI and NotI are underlined), followed byligation into the pET-21a(+) vector (Merck, Darmstadt, Germany)for heterologous expression in Escherichia coli. The resultant re-combinant protein was designed to have a His-tag (AAA-LEHHHHHH) at the C-terminus.

2.2. Protein expression and purification

The E. coli strain BL21 (DE3) was transformed using theobtained plasmid. The transformants were grown in 1 L Luria broth(LB) medium containing 50 lg/ml ampicillin at 37 �C until the cul-ture reached an optical density of 0.6 measured at 600 nm. Theprotein expression was induced with isopropyl-b-D-thiogalactopy-ranoside at a final concentration of 0.2 mM for 24 h at 18 �C. Thecells were harvested and resuspended in 20 ml of 20 mM Tris–HCl buffer (pH 7.5) followed by sonication for 10 min on ice. Aftercentrifugation to remove insoluble material, the supernatant wasapplied onto a nickel (Ni2+) nitrilotriacetic acid (Ni–NTA) agarose(QIAGEN, Hilden, Germany) column equilibrated with the samebuffer. The column was washed with the same buffer, and the re-combinant protein was eluted with the same buffer containing50 mM imidazole. The protein fraction was dialyzed against10 mM Tris–HCl buffer (pH 7.5), and the purity of the eluted pro-tein was analyzed by SDS–PAGE. Selenomethionine (SeMet) deriv-atives were prepared by expressing the protein in the E. coli strainB834 (DE3) grown in LeMaster medium [9]. The SeMet-substitutedprotein was purified in a manner identical to the purification of thenative protein.

2.3. Crystallization, data collection, and structure determination

Prior to crystallization, the recombinant CcGH131A was con-centrated to 9.0 to 12.6 mg/ml in 10 mM Tris–HCl (pH 7.5) usinga Spin-X UF filter unit (Corning, Corning, NY, USA). Needle-shapecrystals of the SeMet-substituted proteins were obtained at 20 �Cusing the hanging-drop vapor diffusion method, in which 1.0 llprotein solution was mixed with an equal volume of the crystalli-zation reservoir solution. Crystal form 1 was obtained using a solu-tion containing 16–20% (wt/wt) polyethylene glycol 4000 (WakoPure Chemical Industries, Osaka, Japan), 50 mM sodium bromide,and 100 mM sodium cacodylate (pH 7.0). Crystal form 2 was grownusing a solution comprising 7% (wt/wt) polyethylene glycol 4000,100 mM ammonium sulfate, and 100 mM sodium phosphate (pH6.0) supplemented with microseeds. The microseeds were pre-pared by crushing the crystals obtained using a solution containing12% (wt/wt) polyethylene glycol 6000 (Hampton Research, AlisoViejo, CA, USA) and 200 mM sodium citrate (pH 5.0). The harvestedcrystals were cryo-protected in a crystallization solution supple-mented with 20% (vol/vol) glycerol and flash-frozen in liquid nitro-gen. The diffraction data sets of SeMet CcGH131A were collected atPF-AR NW12 beamline (Photon Factory, Tsukuba, Japan). All datawere processed and scaled using HKL2000 [10]. Selenium atompositions were determined, and initial phases were calculated fromthe single-wavelength anomalous dispersion data set of the Crystalform 1 (P21) by using the AutoSol program in the PHENIX suite[11]. A rough model of CcGH131A was obtained and further builtusing ARP/wARP [12]. The structure of the Crystal form 2 (P1)was solved by applying the molecular replacement method withMOLREP [13] in the CCP4 program suite using the rough modelof the P21 crystal as a search model. The model was refined usingREFMAC5 in the CCP4 suite [14], and manual adjustment andrebuilding of the model were carried out using the program COOT[15]. Solvent molecules were introduced using the program ARP/wARP. Validation of the structures was performed using MolProbi-ty [16]. In the Ramachandran plot, 96.4% of the residues were inthe favored region, and no residues were identified as outliers.

Figures were prepared using PyMOL (http://www.pymol.org/).The data collection and refinement statistics are summarized inTable 1. The coordinate and structure factors of CcGH131A havebeen deposited in the Protein Data Bank (PDB) under the accessioncodes 3W9A.

3. Results and discussion

3.1. Overall structure

The N-terminal domain of SeMet-substituted CcGH131A (here-after referred to as CcGH131A) produced in E. coli was purified andcrystallized. Two forms of crystals, Crystal forms 1 and 2, which be-long to space groups P21 and P1 were obtained, and they containedtwo molecules and four molecules, respectively, of CcGH131A inthe asymmetric unit. A rough model of the P21 crystal was initiallybuilt at a 2.4-Å resolution, and the structure was further refinedusing the P1 crystal at a 2.0-Å resolution (Table 1). The electrondensity (2Fo–Fc) maps for the four CcGH131A molecules (Mol-Ato Mol-D) contoured at 1 r showed continuous density for almostall main chain atoms except for approximately the last 10 C-termi-nal residues containing the His-tag sequence. The followingdescriptions are based primarily on Mol-A.

The structure of CcGH131A is composed of a b-jelly roll fold(Fig. 1A). Two b-sheets, sheet-A (strands A1–A9) and sheet-B(strands B1–B7), one medium-length a-helix (H1), and two shorta-helices (H2 and H3) are found in the structure. The strand A7is divided into strands A7a and A7b. Structural homology was ana-lyzed using the Dali server [16], and CcGH131A was found to mostresemble endo-b-1,4-glucuronan lyase of the polysaccharide lyase

A

B C

A1A2

A3

A4A5A6

A7aA7b

A8A9

B1

B2

B3

B4B5

B6

B7

H1H2

H3

N

C

Gol-A

Gol-B

Gol-C

90º

Fig. 1. Overall structure of the catalytic domain of CcGH131A. (A) Ribbon model of the catalytic domain of CcGH131A (PDB 3W9A). Sheet-A (A1–A9), Sheet-B (B1–B9), andthree a-helices (H1–H3) are shown in magenta, cyan, and orange, respectively. Three glycerol molecules are shown by yellow stick models. (B) Superimposition of the Cabackbones of CcGH131A (cyan), PL20 Trichoderma reesei endo-b-1,4-glucuronan lyase (PDB 2ZZJ; green), PL13 Bacteroides thetaiotaomicron heparin lyase I (PDB 3INA;magenta), and GH16 Paenibacillus macerans endo-b-1,3-1,4-glucanase (PDB 1U0A; orange). The glycerol molecules bound to CcGH131A are shown in black. (C) Fo–Fc omitmaps for the three glycerol molecules, Gol-A, Gol-B and Gol-C, in the concave of Sheet-B of Mol-A. The maps were calculated by exclusion of the ligands and were contoured at2.0 r.

T. Miyazaki et al. / FEBS Letters 587 (2013) 2193–2198 2195

family (PL) 20 (PDB 2ZZJ; Z score = 16.5) [17]. Interestingly,CcGH131A exhibits a significant similarity to a cellulose-inducedprotein, Cip1, obtained from Trichoderma reesei (PDB 3ZYP; Zscore = 14.7). CcGH131A also shows similarity to the PL13 heparinlyase [18], PL7 alginate lyase [19,20]/polyguluronate lyase [21] (Zscores �13–15), and GH16 b-1,3-1,4-glucanase [22] (Z scores�11–12), all of which consist of b-jelly roll folds.

The active sites of the enzymes that belong to the familiesdescribed above are located in a concave formed by sheet-B. TheCa backbones of CcGH131A and the b-jelly roll fold enzymes aresuperimposed (Fig. 1B). In some of the enzymes, the b-strands ofsheet-B are long, forming a tunnel structure in their active sites.The b-strands of sheet-B of CcGH131A are shorter than those ofother enzymes, which results in a wide and shallow active site ofCcGH131A that may be favorable for the binding of various b-glu-can polysaccharides.

3.2. Active site

Electron density of the three glycerol molecules was observedin the concave of sheet-B of CcGH131A (Fig. 1C). Although non-crystallographic symmetry restraints were not applied to the threeglycerol molecules throughout the refinement process, theyformed the same contacts with CcGH131A in all the four copiesin the asymmetric unit. The glycerol molecules were found to lie

on a straight line and were designated as Gol-A, Gol-B, and Gol-C. The interaction between CcGH131A and the glycerol moleculeswas analyzed using the programs Coot and Ligplot (Fig. 2A andB). The catalytic amino acid residues of glycosidases are typicallyidentified as a pair of carboxylic acid residues [23]. Although theexistence of such a pair was not observed in the residues shownin Fig. 2A, a side chain oxygen atom OE1 of a carboxylic acid resi-due, Glu138, formed hydrogen bonds with oxygen atoms of thetwo glycerol molecules, Gol-B and Gol-C.

Heparin lyase I from Bacteroides thetaiotaomicron (BtHepI) isone of the most structurally homologous enzymes to CcGH131A,and additionally, its catalytic mechanism has been extensivelystudied [18]. To elucidate the catalytic mechanism of CcGH131A,the structures of CcGH131A and BtHepI complexed with a ligand,dodecasaccharide heparin (PDB 3INA), were superimposed. The re-sults suggested a good overlap between the positions of the threeglycerol molecules bound to CcGH131A and the ligand bound toBtHepI (Fig. 2C). The catalytic residues of BtHepI were identifiedas His151 and Tyr357, which function as a proton abstractor anda general acid, respectively [18]. In CcGH131A, Glu138 was foundin the equivalent position of BtHepI His151. In the vicinity of theposition corresponding to Tyr357 in BtHepI, CcGH131A containedArg96 and His218. Atom NH2 of Arg96 formed a hydrogen bondwith atom O3 of Gol-B, and atom NE2 of His218 formed a hydrogenbond with atom O3 of Gol-C; therefore, both residues are likely to

Gol-CGol-A Gol-B

His140 Glu138

Ile222Leu224

Arg96

His218

Tyr51

Gol-CGol-A Gol-B

His140 Glu138

Ile222Leu224

Arg96

His218

Tyr51

231nlG231nlG

09prT09prT

Leu222

His218

Glu98Gln133

His140

Ile224

Glu138Tyr51

Trp90

Arg96

Gol-A

Gol-B

Gol-C

A

B

C

His140Glu138/His151

Arg96

His218

Tyr51Gln132

Tyr357 Glu98

+1 +2-1

-2-3

Gol-C

Gol-B

Gol-A

His140Glu138/His151

Arg96

His218

Tyr51Gln132

Tyr357 Glu98

+1 +2-1

-2-3

Gol-C

Gol-B

Gol-A

Fig. 2. Active site of CcGH131A. (A) Stereo view of residues binding to the glycerol molecules in the concave of the b-jelly roll. Residues forming hydrogen bonds (cyan) andhydrophobic interactions (green) with the ligands (yellow) are shown as stick models. A water molecule and hydrogen bonds are represented by a red sphere and blackdashed lines, respectively. (B) Schematic drawing of the amino acid residues interacting with the ligands. White circle, oxygen atom; black circle, carbon atom; gray circle,nitrogen atom; dashed line, hydrogen bond. Residues involved in hydrophobic interactions are illustrated. (C) Superimposition with CcGH131A (cyan) and BtHepI complexedwith dodecasaccharide (magenta). The catalytic residues of BtHepI and five residues of the dodecasaccharide were derived from the coordinates of 3IMN and 3INA,respectively.

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be important for hydrolysis. It is noteworthy that Arg96 andHis218 also interacted with Glu98 via hydrogen bonds. Alignmentwith other GH131 proteins, such as PaGluc131A, Neurospora crassacellulose-induced secretory protein NCU09764 [24–26], andAspergillus niger An08g04630 [27] was also performed. The fourresidues, Arg96, Glu98, Glu138, and His218 are highly conservedamong GH131 proteins (Fig. 3). Our results suggest that theseresidues may be catalytically critical, and it is likely that the cata-lytic mechanism of CcGH131A is different from that of typical gly-cosidases, which possess a pair of carboxylic acid residues as thecatalytic residues.

An unusual catalytic property referring to a histidine residueacting as a catalytic residue during hydrolysis has been reported

in the structure of two enzymes: a GH3 enzyme, Bacillus subtilisN-acetylglucosaminidase [28] and a GH117 enzyme, Bacteroidesplebeius a-agarase [29]. B. subtilis N-acetylglucosaminidase hasbeen reported to operate by a retaining mechanism, and His324acts as both an acid and a base. In contrast, B. plebeius a-agaraseemploys an inverting mechanism, and His302 acts only as an acid.Regardless of the mechanism, in both the enzymes, an aspartic acidresidue (Asp232 and Asp320, respectively) is located close to thehistidine residue to form a catalytic Asp-His dyad. In the case ofCcGH131A, Glu98 and His218 may form a catalytic dyad, andGlu98 can activate His218 during catalysis. Further studies suchas activity measurement, co-crystallization of wild-type enzymewith inhibitors, and mutational analysis and co-crystallization of

Fig. 3. Sequence alignment of the catalytic domains of GH131 proteins. The alignment was carried out with ClustalW2 [30] using sequences of the GH131 domains fromCoprinopsis cinerea (CcGH131A; Genbank EAU86087.1), Podospora anserina (PaGluc131A; AFQ89876.1), Neurospora crassa (NCU09764; EAA29112.1), and Aspergillus niger(An08g04630; CAK45436.1). Symbols are defined as follows: magenta and blue arrows, Sheet-A and Sheet-B, respectively; orange dotted lines, a-helices; white letters inblack-filled boxes, fully conserved residues among these proteins; letters with gray shadow, identical residues in three sequences; letters in red boxes, residues binding theglycerols.

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the inactive variants with substrate are needed to clarify the struc-ture–function relationship of the GH131 enzymes.

Acknowledgements

We thank Tomoko Fujii for technical assistance. This work wassupported in part by a Grant-in-Aid for Scientific Research (T.T., No.23570132; T.M., No. 25-7279) and a Research Fellowship (T.M.) ofthe Japan Society for the Promotion of Science. This research wasapproved by the Photon Factory Advisory Committee, the NationalLaboratory for High Energy Physics, Tsukuba, Japan (2010G001 and2012G006).

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