Structural basis for entropy-driven cellulose bindingby a type-A cellulose-binding module (CBM)and bacterial expansinNikolaos Georgelisa,1, Neela H. Yennawarb,1, and Daniel J. Cosgrovea,2

aDepartment of Biology, and bHuck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802

Contributed by Daniel J. Cosgrove, August 1, 2012 (sent for review June 29, 2012)

Components of modular cellulases, type-A cellulose-binding mod-ules (CBMs) bind to crystalline cellulose and enhance enzymeeffectiveness, but structural details of the interaction are uncertain.We analyzed cellulose binding by EXLX1, a bacterial expansin withability to loosen plant cell walls and whose domain D2 has type-ACBM characteristics. EXLX1 strongly binds to crystalline cellulosevia D2, whereas its affinity for soluble cellooligosaccharides isweak. Calorimetry indicated cellulose binding was largely entropi-cally driven. We solved the crystal structures of EXLX1 complexedwith cellulose-like oligosaccharides to find that EXLX1 binds theligands through hydrophobic interactions of three linearly arrangedaromatic residues in D2. The crystal structures revealed a uniqueform of ligand-mediated dimerization, with the oligosaccharidesandwiched between two D2 domains in opposite polarity. Thisreport clarifies the molecular target of expansin and the specificmolecular interactions of a type-A CBM with cellulose.

plant cell wall | X-ray crystallography

Gigatons of cellulose are synthesized annually by plants, whichuse this glucose-based polymer as an inert reinforcing ma-

terial in their cell walls. As a combustible fuel and in the form oftextiles, paper, lumber, and other materials, cellulose has vast eco-nomic value, prompting great interest in modifying it by enzymesor other means, particularly for conversion to liquid transpor-tation fuels (1, 2). Cellulose is synthesized as micrometer-longmicrofibrils with ∼24 β- (1, 4)-D-glucans tightly packed into a cable-like array ∼3 nm wide, containing both crystalline and less-orderedregions (3). The crystalline regions have four faces in which eitherthe hydroxyl-rich hydrophilic edges of the glucans or the hydro-phobic pyranose rings populate surfaces of distinctive character.Cellulolytic enzymes typically consist of a catalytic domain linkedto a cellulose-binding module (CBM) that potentiates enzyme ac-tivity by proximity and targeting effects (4–6). Type-A CBMs use aflat surface populated with aromatic residues to bind to crystallinecellulose whereas type-B CBMs use a deep groove to bind indi-vidual twisted glucan chains found in disordered cellulose (4, 7).Although there are numerous published structures of type-B

CBMs complexed with cellooligosaccharides (8–12), analogousstructures for type-A CBMs have proved difficult to produce, andso the current concept of type-A CBM interaction with celluloseis based on indirect evidence. The current hypothesis, that type-ACBMs bind to the hydrophobic face of crystalline cellulose, stemsfrom the observation that they have an open surface with a planarstrip of aromatic residues suitably positioned to bind the planarsurface of the aligned pyranose rings in crystalline cellulose(4, 13). Site-directed mutagenesis showed these aromatic residuesto be essential for binding crystalline cellulose (14–17), andcalorimetry indicated binding was mainly driven by increases inentropy, consistent with freeing of constrained water at the hy-drophobic surfaces of the protein and cellulose (18). Supportingthis idea, electron microscopy indicated that type-A CBMs bindto the hydrophobic face of crystalline cellulose (19). However,structural details of the interaction of type-A CBMs with celluloseare lacking because suitable protein:ligand complexes have not been

crystallized to date. Such structures could help elucidate how type-A CBMs may modify cellulose (20, 21) and potentiate cellulaseaction (4–6, 22), with consequences for the engineering andtechnology of cellulose use as a material and as a feedstock forbiofuel production.Here we report energetic and structural details of cellulose

binding by a type-ACBM that is a tightly integrated part of EXLX1,a bacterial expansin from Bacillus subtilis (23, 24). Expansins werefirst discovered as nonenzymatic plant proteins that loosen plantcell walls (25), with key roles in plant cell growth and develop-mental processes that involve cell-wall modification, such as leafabscission, fruit softening, and pollen tube penetration of thestigma (26, 27). Bacterial expansins were recently recognized byvirtue of their structural similarity to plant expansins (23, 28). Theyfacilitate microbial colonization of plants, with consequences forpathogen virulence and biocontrol (23, 29).We focused on EXLX1 because it is readily expressed in active

form in heterologous expression systems, whereas plant expan-sins have proved recalcitrant to this approach. As is true forother expansins, domain D1 of EXLX1 shows distant structuralsimilarity to family-45 glycoside hydrolases, but has no detectablelytic activity against the polysaccharide components of plant cellwalls (23). Domain D2 forms an open, nearly flat surface resem-bling that of type-A CBMs (23, 24). Detailed analysis of EXLX1indicated that both domains are essential for plant cell-wall loos-ening activity and domain D2 almost exclusively mediates bindingto cellulose (24).In this study, we analyzed EXLX1 binding to cellulose and

assessed whether it competes for binding sites with type-A ortype-B CBMs. Additionally, we obtained calorimetric and crys-tallographic data to reveal the thermodynamics and physical na-ture of EXLX1 binding to cellulose. Our results provide insightinto the specific target of EXLX1 and, by extension, of other ex-pansins and type-A CBMs.

ResultsEXLX1 Binding to Cellulose. The predominant forms of celluloseused to characterize CBM binding are, in decreasing degree ofcrystallinity, bacterial microcrystalline cellulose (BMCC), Avicel,and phosphoric-acid–swollen cellulose (PASC) (30–32). BMCCand Avicel have higher binding capacity for type-A CBMs com-pared with type-B and have been used to distinguish between the

Author contributions: N.G., N.H.Y., and D.J.C. designed research; N.G. and N.H.Y. per-formed research; N.G., N.H.Y., and D.J.C. analyzed data; and N.G., N.H.Y., and D.J.C. wrotethe paper.

The authors declare no conflict of interest.

Data deposition: The crystallography, atomic coordinates, and structure factors havebeen deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4FER, 4FG2,4FFT, and 4Fg4).1N.G. and N.H.Y. contributed equally to this work.2To whom correspondence should be addressed. E-mail: dcosgrove@psu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1213200109/-/DCSupplemental.

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two groups of CBMs, whereas PASC has comparable bindingcapacities for the two types (14, 32–35).We compared EXLX1 binding to cellulose with that of two

type-A CBMs (CBM3 and CBM10) and two type-B CBMs(CBM17 and CBM28). The purity of all proteins was checked bySDS/PAGE (Fig. S1). Protein binding to Avicel and BMCC wasfitted to a Langmuir-type model as described before (14, 18, 36).Binding data approximate a one-site Langmuir isotherm up toprotein concentrations of 20–25 μM (Fig. S2). At higher concen-trations, there was evidence of a second binding site of much loweraffinity, which was observed previously for type-A and type-B CBMs(18, 34). For simplicity, we ignored this site because its relativecontribution to binding was negligible at the protein concentra-tions used here. Binding to BMCC resembled that of type-A CBMs,although with fewer binding sites, and for Avicel its binding prop-erties were intermediate between the two types of CBMs (Table 1),with a dissociation constant Kd ∼2 μM.Although cellulose binding for most CBMs is reversible, there

are examples of CBMs that bind to crystalline cellulose irreversibly(14, 19, 37), which can complicate the analysis. We found thatEXLX1 binds Avicel and BMCC reversibly (assayed at low con-centrations, [EXLX1]free ≤ Kd; Fig. S3).

Binding Competition Between EXLX1 and CBMs. To assess overlap inbinding sites, we measured EXLX1 binding to Avicel ± a fixedamount of a type-A or type-B CBM. We used Avicel because,unlike BMCC, it contained binding sites for both types of CBMs.The CBM amount was enough to occupy 80–85% of the corre-sponding binding sites. Type-A, but not type-B, CBMs substan-tially reduced EXLX1 binding (Fig. 1). BSA (a negative control)did not interfere with EXLX1 binding. These results show thatthe cellulose surfaces recognized by EXLX1 overlap, at leastpartially, with those of type-A CBMs.

Specificity and Thermodynamics of EXLX1 Binding.We tested EXLX1binding to various soluble plant cell-wall components by iso-thermal titration calorimetry (ITC), which can be used to calculatethe thermodynamic parameters (ΔH, ΔS, and ΔG), Kd, andstoichiometry of binding. Most glycans that we tested (cello-pentaose, xyloglucan hepta-oligosaccharide, xylohexaose, man-nohexaose, arabinan, galactan, arabinoxylan, and xyloglucan)resulted in no heat change upon mixing with EXLX1 in solution,other than the heat of dilution. Cellohexaose and mixed-linkage β-(1, 3) (1, 4)-D-glucan (MLG) gave small heat releases, with agradual reduction as more glycan was added, indicating weakbinding by EXLX1 (Kd > 1 mM) (Fig. S4). Alanine substitutions

of W125, W126, and Y157 in the triple EXLX1 variant TTTeliminated this heat release, demonstrating their importance inbinding of these ligands (Fig. S4). These aromatic residues form aplanar surface in D2 (similar to type-A CBMs) and have beenimplicated in cellulose binding and cell-wall loosening activity (24).The tight binding of EXLX1 to crystalline cellulose (Kd ∼2.0 μM)and weak affinity for soluble cellooligosaccharides (Kd > 1.0 mM)are also features of type-A CBMs (4, 15). In contrast, type-B CBMs,such as CBM17, show high affinity for soluble cellooligosaccharides(33), which was not seen with EXLX1 (Fig. S4).Our results indicate that insoluble cellulose is the primary

binding target of the flat aromatic surface of EXLX1. Our attemptsto measure the thermodynamic parameters of EXLX1 binding toAvicel and BMCC with ITC were unsuccessful because the low-binding capacity of these materials for EXLX1 required the use ofexcessively high amounts of cellulose, resulting in unstable base-lines and undetectable ΔH signal upon EXLX1 injection. In com-parison, PASC showed higher EXLX1-binding capacity (Bmax =3.5 μmol/g versus 0.3 μmol/g for Avicel) (Table 1, Fig. S2), in partbecause of its greater exposed surface area. Still, ΔH for EXLX1binding to PASC was below the ITC detection limit (Fig. 2). Incontrast, PASC binding by type-B CBM17 yielded a large ΔH(−13.3 ± 0.6 kcal mol−1) and entropy decreased (T*ΔS =−5.6 kcal mol−1). Based on these results, we conclude that ΔHof EXLX1 binding to PASC is at least tenfold lower in absolutevalue than the ΔH for CBM17 binding to PASC (that is,jΔHj<1.3 kcal mol−1). Therefore, the change in free energyassociated with EXLX1 binding to PASC, ΔG = −RTlnKa =

Table 1. Characterization of the binding of EXLX1 (including Alexa-labeled EXLX1) and variousCBMs to Avicel and BMCC

Substrate ProteinBmax (μmol/g ofsubstrate ± SEM) Kd (μM ± SEM)

BMCC CBM3 (type-A) 9.9 ± 0.6 0.5 ± 0.2CBM10 (type-A) 6.4 ± 0.2 4.2 ± 0.3EXLX1 1.0 ± 0.1 1.9 ± 0.4CBM17 (type-B) Not detected Not detectedCBM28 (type-B) Not detected Not detected

Avicel CBM10 (type-A) 2.7 ± 0.2 6.7 ± 0.8CBM3 (type-A) 0.6 ± 0.1 0.7 ± 0.2EXLX1 0.3 ± 0.0 2.3 ± 0.2CBM17 (type-B) 0.3 ± 0.0 2.1 ± 0.4CBM28 (type-B) 0.1 ± 0.0 3.7 ± 1.0Alexa-EXLX1 0.3 ± 0.0 2.7 ± 0.5

PASC EXLX1 3.5 ± 0.1 1.0 ± 0.1CBM17 (type-B) 9.7 ± 1.0 1.2 ± 0.5

Hepes (50 mM) pH 7.5, 5 mM CaCl2, 25 mM NaCl was used as a buffer.

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4.4 ±1.1 (CBM17)

9.6 ± 1.3 † (EXLX1)

2.3 ± 0.2 (No comp.) 2.3 ± 0.2 (No comp.)

Fig. 1. Lineweaver-Burk plots of Alexa-labeled EXLX1 binding to Avicel inthe presence of nonlabeled EXLX1, type-A CBMs (17 μMCBM3, 40 μMCBM10)and type-B CBMs (20 μM CBM17, 20 μM CBM28). BSA was used as a negativecontrol. The values represent the apparent affinity of EXLX1 for Avicel (μM ±SEM) in the presence of the competitor in parenthesis. † P < 0.01 comparedwith binding of EXLX1 in the absence of a competitor (n = 5, F test). Nocomp.: No competitor.

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−8.2 kcal mol−1 (Fig. 2), is mostly the result of increased entropy(ΔG = ΔH-T*ΔS, T: temperature in Kelvin).

Crystal Structure of the EXLX1–Cellohexaose Complex. We producedcrystals of EXLX1 complexed with cellohexaose (the hexamericcellooligosaccharide) to gain insight into the interaction of EXLX1with cellulose. The structure was solved and refined to 2.1-Åresolution with a crystallographic R factor of 0.172 and an R free of0.201 (Table S1).Analysis of the structure revealed a unique protein:ligand ar-

rangement. One molecule of cellohexaose is packed between thearomatic surfaces of domain D2 of two EXLX1 molecules, desig-nated (A) and (B), with the reducing end of cellohexaose pointingtoward the D1 domain of EXLX1(B) (Fig. 3A). Dynamic lightscattering of EXLX1 in solution showed that EXLX1 is a mono-mer (size estimated as 19 kDa) both in the absence and presenceof cellohexaose, indicating dimerization by cellohexaose does notoccur in solution, at least under the conditions tested.The structures of EXLX1(A) and (B) are essentially identical

to each other and to the existing crystal structure of EXLX1(PDB#: 3D30), which was obtained without bound ligands[rmsd = 0.443 and 0.491 Å for superposition of 3D30 withEXLX1(A) and EXLX1(B) respectively] (Fig. S5). There is nochange in the position of the side chains of amino acid residuesthat are crucial for cellulose and cell-wall–modifying activity,including D71, Y73, D82, K119, W125, W126, and Y157 (24).The only minor change is that W125 is slightly tilted in bothEXLX1 monomers compared with 3D30 (Fig. S5).Binding of CBMs to cellulose is partly mediated by hydro-

phobic interactions between aromatic amino acids and pyranoserings (4). Consistent with such a mechanism, the aromatic ringsof W125, W126, and Y157 in both EXLX1(A) and EXLX1(B)are nearly parallel to the planes of the pyranose rings of cello-hexaose, which adopt the undistorted 4C1 chair conformation.The distance between the aromatic and the pyranose rings is 3.0–4.0 Å, allowing CH–π interactions (38, 39). In EXLX1(A), W126makes CH–π interactions with glucose (G) residue G4 (numberingof pyranose rings starts from the reducing end of cellohexaose),and W125 interacts with G2 and to a lesser extent with G1 (Fig.3B). The aromatic ring of Y157 is located between G5 and G6 (5.1and 5.3 Å from ring centers, respectively), likely interacting withboth. In EXLX1(B), W125 is stacked against G5 andW126 makes

CH–π interactions with G3 and to a lesser extent with G2 (Fig.3B). Similarly, Y157 in EXLX1(B) is located between two pyranoserings, G1 and G2, 5.0 and 5.9 Å from the ring centers (Fig. 3B).The distances between W125, W126, and Y157 do not match

the 5.5-Å interval between pyranose rings of cellohexaose and soit is not possible for all aromatic amino acids to overlap preciselywith pyranose rings at the same time (Fig. 4). In both EXLX1(A)and EXLX1(B), W125 and W126 overlap better with pyranoserings than does Y157 (Figs. 3B and 4), suggesting that theseresidues are more important for cellulose binding than Y157.Supporting this idea, the single amino acid variants W125A andW126A bound cellulose less effectively than wild-type EXLX1,whereas the Y157A variant was less affected (24).There are few direct H bonds between cellohexaose and EXLX1,

formed only by K119 (Fig. 3B). In EXLX(A), K119 forms Hbonds with the C2 and C3 hydroxyl groups of G5; whereas, inEXLX(B) it is hydrogen bonded with the C3 hydroxyl of G1 andthe C6 hydroxyl of G2 as well as with the G1:G2 glycosidic ox-ygen. The direct contact of K119 with cellohexaose is in agree-ment with mutagenesis data implicating K119 in cellulose bindingand cell-wall loosening (24). Indirect H bonding via water can beseen but contribute little to CBM binding (40).

Structures of EXLX1 with Cellotetraose, Hemithiocellodextrin, andMLG. In the EXLX1:cellohexaose structure, domain D1 did notcontact cellohexaose, possibly because of its weak affinity forcellulose (24). In attempts to obtain complexes where the glucansinteract directly with residues in D1, we successfully produced

0.0 0.5 1.0 1.5 2.0

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Fig. 2. Binding interaction between EXLX1 and PASC as investigated byisothermal titration calorimetry. The binding parameters are indicated in-side each graph. The interaction of CBM17 with PASC was used as positivecontrol. *ΔG was calculated from the Kd value in Table 1. The experimentwas repeated three times with similar results.

D1

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G1 G2 G3 G4 G5 G6

K119Y157

W126W125

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Fig. 3. Structure of EXLX1 with cellohexaose. (A) Sandwich structure con-sisting of two molecules of EXLX1 (A and B) and one molecule of cellohex-aose (green). (B) Direct hydrogen bonds and CH–π interactions between eachEXLX1 molecule and cellohexaose. Glucose numbering starts from the re-ducing end of cellohexaose.

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crystals of EXLX1with cellotetraose, hemithiocellodextrin (HTC;DP = 10) and oligosaccharides from MLG, with the last twoligands being longer than cellohexaose. HTC is a water-solubleanalog of cellodecaose (water insoluble), except that every otherglycosidic linkage is replaced with a thio-glycosidic linkage (41).We hoped that use of long oligosaccharides such as HTC andMLG would result in EXLX1 structures where the ligands span-ned both EXLX1 domains.In all three of these structures, the ligands bind to EXLX1 in a

fashion similar to that found for cellohexaose (Fig. S6). Theinteractions of D2 with all three ligands are dominated by CH–πinteractions mediated by aromatic amino acids W125, W126,and Y157 with minimal contribution by direct H bonding.Sandwich structures formed in all these cases, with the ligandbound between the aromatic surfaces of D2 domains of twoEXLX1 in opposite polarity. Thus, this sandwich structureseems to be a thermodynamically favorable configuration withvarious cellulose-like oligosaccharides.These structures also show that domain D1 does not contrib-

ute to ligand binding in these complexes. This suggests that D1binding to glucans is extremely weak, consistent with bindingresults (24), and thus domain D1 may depend on D2 bindingto cellulose for performing its loosening action on celluloseand cell-wall networks.

DiscussionEXLX1 domain D2 has characteristics of a type-A CBM. Ourstructures of EXLX1 with cellohexaose and other oligosaccharidesprovide the first structures of a type-A CBM in complex with acellulose-like oligosaccharide and reveal the structural details oftype-A CBM binding to cellulose. Although cellohexaose is moreflexible in solution than are the glucan chains in crystalline cel-lulose, it is the closest water-soluble equivalent to crystalline cel-lulose. Therefore, lessons learned from the structure of EXLX1complexed with cellohexaose can provide information about thebinding of EXLX1 (and potentially other type-A CBMs and ex-pansins) to cellulose.Our results show that EXLX1 binding to cellohexaose and

related oligosaccharides is dominated by CH–π interactions ofW125, W126, and Y157 with the glucan’s pyranose rings, in aconfiguration similar to that hypothesized for type-A CBMs. Theregistration of these aromatic residues with the pyranose rings is

shown in Fig. 4, which also illustrates the spacing of analogousaromatic residues from the binding surfaces of four other type-ACBMs. The number and general arrangement of the aromaticresidues are similar among the protein surfaces, but the spacingshows substantial variability in the specific geometry of thebinding interactions.ITC showed that ΔH for EXLX1 binding to cellulose is a small

component of ΔG of binding (< 16%), indicating that an in-crease in entropy drives binding, mostly likely due to release ofwater constrained to unfavorable configurations at the hydro-phobic surfaces of cellulose and protein. Similarly, the binding oftype-A CBMs to cellulose is believed to be driven by positiveentropic changes due to the release of bound water (18). Thebinding affinities of D2 and other type-A CBMs to soluble cel-looligosaccharides are ∼1,000-fold lower than to cellulose, pos-sibly because of the loss in entropy when the cellooligosaccharideslose conformational freedom and the increase in enthalpy whenthey are forced into an energetically unfavorable conformation,whereas these thermodynamic penalties are not incurred whenthe protein binds to an already constrained glucan on the cellulosesurface. A comparison among cellooligosaccharide conformationsshows that cellohexaose bound to EXLX1 is intermediate betweenthe flat chains in cellulose and the twisted chains in type-B CBMcomplexes (Fig. S7). These observations are consistent with thecompetition results of Fig. 1 and indicate that EXLX1 may bindto the more ordered regions of cellulose rather than to the dis-ordered regions targeted by type-B CBMs.One particularly intriguing observation in our study is that two

molecules of EXLX1 form a sandwich structure with one oli-gosaccharide between the aromatic surfaces to two D2 domains.This configuration appears to be unique, yet it occurred with allfour oligosaccharides that formed an EXLX1 crystal complex. Asecond unique observation is that the two D2 surfaces bind theoligosaccharides in opposite polarities with respect to the reducingend of the glucan chain. This likewise seems remarkable. Theclosest comparison may be found in the ligand-mediated dimerof the E78R mutant of the type-B CMB, PeCBM29-2 (42), butin this structure the two proteins bind the oligosaccharide in thesame polarity and the oligosaccharide is found in the twistedconfiguration characteristic of type-B CBMs. Furthermore, thetwo PeCBM29-2 proteins make numerous direct H bonds witheach other that stabilize the dimer, whereas such interactions arenot seen in the EXLX1:oligosaccharide structures, where the twoproteins are connected only via the oligosaccharide.The unusual binding of EXLX1 to cellulose-like oligosaccharides

in opposing polarities may be possible because similar and relativelynonspecific CH–π interactions are made by both proteins on opensurfaces. In addition, binding in opposite polarities could beenabled by the scarcity of direct H bonds, which require the -OHgroups to be in specific positions and orientations. In nature,most -OH groups in crystalline cellulose would be H bonded toadjacent glucose residues in the same plane, thus limiting theiravailability for binding with surface proteins. This aspect ofEXLX1 binding may influence the dynamics of its movement oncellulose surfaces and may be common in type-A CBMs, one ofwhich has been shown to diffuse along the surface of cellulose,although it is not clear whether it displays movement anisotropyalong a single microfibril (43) as has been observed for cello-biohydrolase I (Cel7A) (44). Our results suggest that EXLX1could move bidirectionally on a microfibril.The exact target of expansin has been a subject of interest and

speculation in an effort to understand the site and mechanism ofits wall-loosening action (26, 45). Our results indicate that EXLX1shares binding sites with type-A CBM3, reported to be located onthe hydrophobic face of cellulose microfibrils (19). These results,along with the arrangement of aromatic residues on the D2 sur-face and the hydrophobic nature of D2 binding to cellooligosac-charides, collectively indicate that EXLX1 binds to the hydro-

EXLX1

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Fig. 4. Alignment and registration of aromatic residues of EXLX1 with thepyranose rings of cellohexaose in the protein:ligand complex (Left) and com-parison with the spacing of analogous surface aromatic residues in four othertype-A CBMs. PDB numbers of the structures are indicated in parentheses.

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phobic face of the cellulose microfibril. However, it is unlikelythat EXLX1 binds to the whole hydrophobic face because thebinding capacity of the highly crystalline BMCC for EXLX1 isfive- to tenfold lower than for CBM3 and CBM10. In addition,there is a slight right-handed twist among aromatic residues W125,W126, and Y157 (Fig. 3 and Fig. S5). This slight twist, which ismirrored in the EXLX1-bound cellohexaose (Fig. S7), may pref-erentially direct binding to cellulose surfaces with a similar twist.These sites might be junctions between crystalline and disorderedcellulose, perhaps similar to the proposed targets of endogluca-nase CtCel124, which has distinct binding surfaces for crystallineand noncrystalline cellulose (46). One speculative scenario is thatEXLX1 binds to the slightly twisted region of a glucan chain thatbridges two cellulose microfibrils. Such stray or disordered glucansare important in strengthening paper, a synthetic network of cel-lulose fibers that is weakened by EXLX1 (24). In this scenario, D2binding might assist in unzipping the glucan from the cellulosesurface (with energy provided by turgor-generated wall stresses)and would also lead domain D1 to interact with the disorderedpart of the same glucan chain.Because the binding of expansin domain D1 to cellulose is

extremely weak, we infer that the function of domain D1 is highlydependent on the binding of D2 to cellulose. The weak binding issupported by previous work (24) as well as our current ITC re-sults and the lack of stable interactions between D1 and oligo-saccharides in our four EXLX1:ligand structures. Nonetheless,domain D1 is clearly needed for loosening activity by EXLX1(24). The D1 surface formed by residues D82, D71, and Y73extends the cellulose-binding surface of D2 and has been shownby site-directed mutagenesis to be critical for weakening filter pa-per and for loosening plant cell walls (24). Although expansins lackwall lytic activity, domain D1 shows distant structure similarity toGH45 endoglucanases (23). Cellooligosaccharides do stably bind toGH45 endoglucanases (23, 47) where the catalytic site is located ina deep cleft containing several polar residues that help bind thecellooligosaccharide (47). In contrast, EXLX1 domain D1forms an open surface with a small shoulder near residues D71,D82, and Y73; the surface is much flatter than the one in GH45endoglucanases, missing residues that stabilize the interaction withcellooligosaccharides, as well as missing key parts of the catalyticmachinery (23, 24, 47). Thus, the specific molecular activity of D1that leads to its wall-loosening activity differs from GH45 actionand may not require a stable interaction with the glucan.In summary, our study revealed both predicted and unex-

pected features of EXLX1 binding to cellulose, with conclusionsthat may also hold true for other expansins and type-A CBMs inmodular cellulases. The structure of D2 with cellohexaose can bea starting point for molecular dynamics simulations to better un-derstand how the binding of expansin to cellulose may facilitaterelease of glucans from cellulose microfibrils. Such studies offerthe potential for deeper understanding of the biophysical actionsof expansins on cell-wall networks and type-A CBMs on cellulosesurfaces, with potential applications in many fields.

Materials and MethodsOligosaccharides and Polysaccharides. Avicel PH-101 was purchased from FMCBiopolymer. BMCC was prepared from Gluconacetobacter xylinus pellicles asdescribed by Gilkes et al. (36). PASC was prepared from Avicel PH-101 asdescribed by Wood (48). Cellohexaose, cellopentaose, xylohexaose, man-nohexaose, xyloglucan heptaoligosaccharide (Cat: O-X3G4), sugar beet ara-binan (Cat: P-ARAB), potato galactan (Cat: P-GAPT), barley mixed-linkageglucan (low viscosity, Cat: P-BGBL), wheat arabinoxylan (Cat: P-WAXYL), andtamarind xyloglucan (Cat: P-XYGLN) were purchased from Megazyme. Cel-lotetraose was purchased from Seikagaku Corp.

Protein Expression, Purification, and Labeling. EXLX1 and the TTT variant wasexpressed, purified, and labeled with Alexa Fluor 488 C5-maleimide (Life

Technologies) as described by Georgelis et al. (24). The cDNA sequence ofPseudomonas fluorescens CBM10 (PDB#: 1E8R) (35, 49) was synthesized byLife Technologies and the protein was expressed and purified as describedby Bolam et al. (5). Clostridium cellulovorans CBM17 (PDB#: 1J83) (10, 33, 34)and Bacillus sp.1139 CBM28 (PDB#: 1UWW) (34, 50) were provided byDr. Alisdair Boraston (University of Victoria, Victoria, Canada) in lyophilizedform. C. thermocellum CBM3 (PDB#: 1NBC) (13, 19) was purchased fromProzomix Limited. All proteins were extensively exchanged into 50mMHepes(pH 7.5), 5 mM CaCl2, 25 mM NaCl2 by use of polyethersulfone (PES) con-centrating columns (3kD cutoff) (Sartorius Stedium Biotech). The purity of theproteins was visualized by 15% (wt/vol) SDS/PAGE stained with CoomassieBrilliant Blue R-250. SeeBlue Plus 2 was used as a standard (Life Technologies).

Protein Crystallization, Data Collection, Structure Determination, and Refinement.EXLX1 was concentrated to 30 mg/mL in the presence of cellohexaose orhemithiocellodextrin or MLG or cellotetraose, crystallized by hanging drops,and data were collected at an MM007 rotating anode X-ray generator withCuKα radiation, operating at 1.2 kW of power and Saturn944+ CCD detector(Rigaku Americas). For details see SI Material and Methods.

Isothermal Titration Calorimetry. The thermodynamic parameters of EXLX1binding to soluble oligosaccharides, polysaccharides, and PASC were assessedat 25 °C with an ITC-200 microcalorimeter (MicroCal). EXLX1, CBM17, andPASC were dialyzed in 50 mM Hepes (pH 7.5), 5 mM CaCl2, 25 mM NaCl,0.01% NaN3 for 24 h at room temperature and saccharides were dissolved in50 mM Hepes (pH 7.5), 5 mM CaCl2, 25 mM NaCl, 0.01% NaN3. EXLX1 wasplaced into the ITC-200 cell (202.8 μL working volume) at a final concen-tration of 500 μM. The concentration of CBM17 (positive control) (10, 51) inthe cell was 200 μM. Bindings were analyzed by 4 μL injections of ligandconcentrated to 10 mg/mL. Cellohexaose was concentrated to 3 mg/mL forinjections to a cell containing CBM17. For ITC analysis of the EXLX1 andCBM17 (51) binding to PASC, 10.5 mg/mL PASC (or 36.5 μM of EXLX1binding sites), and 3.5 mg/mL PASC (or 34 μM of CBM17 binding sites)respectively, were placed in the cell. Binding was analyzed by 4-μL injec-tions of EXLX1 or CBM17 concentrated to 500 μM. Data were processed withOrigin 7 (MicroCal).

Cellulose-Binding Assays. Avicel, BMCC, and PASC binding studies of allproteins were done as described by Georgelis et al. (24). The buffer used in allbinding assays was 50 mM Hepes (pH 7.5), 5 mM CaCl2, 25 mM NaCl2 . Thebinding parameters (dissociation constant and binding capacity) were cal-culated by fitting the data to a single-site Langmuir isotherm with GraphpadPrism version 5.04 (GraphPad Software).

Binding Competition Assays. EXLX1 binding to Avicel was studied as describedabove in the presence of a fixed amount of CBM3 (17 μM), CBM10 (40 μM),CBM17 (20 μM), or CBM28 (20 μM). EXLX1 used in these experiments con-tained a proportion of one molecule of Alexa-labeled EXLX1 to 24 non-labeled EXLX1molecules (24). Fixed amounts of BSA (20 μM) and nonlabeledEXLX1 (20 μM) were used as negative and positive control for bindingcompetition, respectively. Binding of EXLX1 to Avicel was assessed by fluo-rescence of EXLX1-attached Alexa Fluor 488 with a NanoDrop 3300 fluo-rospectrometer (Thermo Fisher Scientific). The statistics of the binding ofAlexa-labeled EXLX1 to Avicel in the presence of competing proteins werecalculated with Graphpad Prism 5.04.

Protein Models. Protein structures were visualized with Chimera software(University of California, San Francisco) (52). Potential H bonds were placedon the structures by use of the FindHbond function of Chimera and relaxingH-bond constraints by 0.4 Å and 20°. The structures were deposited in RCSBProtein Data Bank as #4FER, 4FG2, 4FFT, and 4FG4.

Additional Details. See SI Materials and Methods.

ACKNOWLEDGMENTS. We are grateful to Lisa Wilson, Daniel Durachko, andEdward Wagner for technical assistance, Hemant Yennawar (Department ofChemistry, Pennsylvania State University, University Park, PA) for help withX-ray crystallography, Alisdair Boraston (Department of Biochemistry andMicrobiology, University of Victoria, Victoria, Canada) for providing CBM17and CBM28, and Linghao Zhong (Department of Chemistry, PennsylvaniaState University, University Park, PA) for helpful discussion. This work wassupported by United States Department of Energy Grant DE-FG02-84ER13179from the Office of Basic Energy Sciences.

14834 | www.pnas.org/cgi/doi/10.1073/pnas.1213200109 Georgelis et al.

1. Himmel ME, et al. (2007) Biomass recalcitrance: Engineering plants and enzymes forbiofuels production. Science 315:804–807.

2. Rubin EM (2008) Genomics of cellulosic biofuels. Nature 454:841–845.3. Fernandes AN, et al. (2011) Nanostructure of cellulose microfibrils in spruce wood.

Proc Natl Acad Sci USA 108:E1195–E1203.4. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydrate-binding modules:

Fine-tuning polysaccharide recognition. Biochem J 382:769–781.5. Bolam DN, et al. (1998) Pseudomonas cellulose-binding domains mediate their effects

by increasing enzyme substrate proximity. Biochem J 331:775–781.6. Hervé C, et al. (2010) Carbohydrate-binding modules promote the enzymatic de-

construction of intact plant cell walls by targeting and proximity effects. Proc NatlAcad Sci USA 107:15293–15298.

7. Boraston AB, Lammerts van Bureren A, Ficko-Blean E, Abbott DW (2007) Carbohy-drate-protein interactions: Carbohydrate-binding modules. Comprehensive Glyco-science, ed Kamerling JP (Elsevier, Amsterdam), pp 661–696.

8. Tsukimoto K, et al. (2010) Recognition of cellooligosaccharides by a family 28 car-bohydrate-binding module. FEBS Lett 584:1205–1211.

9. Bae B, et al. (2008) Molecular basis for the selectivity and specificity of ligand rec-ognition by the family 16 carbohydrate-binding modules from Thermoanaer-obacterium polysaccharolyticum ManA. J Biol Chem 283:12415–12425.

10. Notenboom V, et al. (2001) Recognition of cello-oligosaccharides by a family 17 car-bohydrate-binding module: An X-ray crystallographic, thermodynamic and muta-genic study. J Mol Biol 314:797–806.

11. Boraston AB, et al. (2002) Differential oligosaccharide recognition by evolutionarily-related beta-1,4 and beta-1,3 glucan-binding modules. J Mol Biol 319:1143–1156.

12. Charnock SJ, et al. (2002) Promiscuity in ligand-binding: The three-dimensionalstructure of a Piromyces carbohydrate-binding module, CBM29-2, in complex withcello- and mannohexaose. Proc Natl Acad Sci USA 99:14077–14082.

13. Tormo J, et al. (1996) Crystal structure of a bacterial family-III cellulose-binding do-main: A general mechanism for attachment to cellulose. EMBO J 15:5739–5751.

14. McLean BW, et al. (2000) Analysis of binding of the family 2a carbohydrate-bindingmodule from Cellulomonas fimi xylanase 10A to cellulose: Specificity and identifica-tion of functionally important amino acid residues. Protein Eng 13:801–809.

15. Nagy T, et al. (1998) All three surface tryptophans in Type IIa cellulose binding do-mains play a pivotal role in binding both soluble and insoluble ligands. FEBS Lett 429:312–316.

16. Linder M, et al. (1995) Identification of functionally important amino acids in thecellulose-binding domain of Trichoderma reesei cellobiohydrolase I. Protein Sci 4:1056–1064.

17. Reinikainen T, Teleman O, Teeri TT (1995) Effects of pH and high ionic strength on theadsorption and activity of native and mutated cellobiohydrolase I from Trichodermareesei. Proteins 22:392–403.

18. Creagh AL, Ong E, Jervis E, Kilburn DG, Haynes CA (1996) Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble micro-crystalline cellulose is entropically driven. Proc Natl Acad Sci USA 93:12229–12234.

19. Lehtiö J, et al. (2003) The binding specificity and affinity determinants of family 1 andfamily 3 cellulose binding modules. Proc Natl Acad Sci USA 100:484–489.

20. Din N, et al. (1994) C1-Cx revisited: Intramolecular synergism in a cellulase. Proc NatlAcad Sci USA 91:11383–11387.

21. Wang L, Zhang Y, Gao P (2008) A novel function for the cellulose binding module ofcellobiohydrolase I. Sci China C Life Sci 51:620–629.

22. Percival Zhang YH, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement:Screening and selection strategies. Biotechnol Adv 24:452–481.

23. Kerff F, et al. (2008) Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1),a bacterial expansin that promotes root colonization. Proc Natl Acad Sci USA 105:16876–16881.

24. Georgelis N, Tabuchi A, Nikolaidis N, Cosgrove DJ (2011) Structure-function analysis ofthe bacterial expansin EXLX1. J Biol Chem 286:16814–16823.

25. McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two endogenous proteins thatinduce cell wall extension in plants. Plant Cell 4:1425–1433.

26. Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6:850–861.27. Tabuchi A, Li LC, Cosgrove DJ (2011) Matrix solubilization and cell wall weakening by

β-expansin (group-1 allergen) from maize pollen. Plant J 68:546–559.28. Yennawar NH, Li LC, Dudzinski DM, Tabuchi A, Cosgrove DJ (2006) Crystal structure

and activities of EXPB1 (Zea m 1), a beta-expansin and group-1 pollen allergen frommaize. Proc Natl Acad Sci USA 103:14664–14671.

29. Jahr H, Dreier J, Meletzus D, Bahro R, Eichenlaub R (2000) The endo-β-1,4-glucanaseCelA of Clavibacter michiganensis subsp. michiganensis is a pathogenicity de-

terminant required for induction of bacterial wilt of tomato. Mol Plant Microbe In-

teract 13:703–714.30. Park S, Johnson DK, Ishizawa CI, Parilla PA, Davis MF (2009) Measuring the crystallinity

index of cellulose by solid state (13)C nuclear magnetic resonance. Cellulose 16:

641–647.31. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS (2010) Cellulose crystallinity—A key

predictor of the enzymatic hydrolysis rate. FEBS J 277:1571–1582.32. McLean BW, et al. (2002) Carbohydrate-binding modules recognize fine substructures

of cellulose. J Biol Chem 277:50245–50254.33. Boraston AB, Chiu P, Warren RA, Kilburn DG (2000) Specificity and affinity of sub-

strate binding by a family 17 carbohydrate-binding module from Clostridium cellu-

lovorans cellulase 5A. Biochemistry 39:11129–11136.34. Boraston AB, Kwan E, Chiu P, Warren RA, Kilburn DG (2003) Recognition and hy-

drolysis of noncrystalline cellulose. J Biol Chem 278:6120–6127.35. Ponyi T, et al. (2000) Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the

family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble li-

gands. Biochemistry 39:985–991.36. Gilkes NR, et al. (1992) The adsorption of a bacterial cellulase and its two isolated

domains to crystalline cellulose. J Biol Chem 267:6743–6749.37. Linder M, Teeri TT (1996) The cellulose-binding domain of the major cellobiohy-

drolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on

crystalline cellulose. Proc Natl Acad Sci USA 93:12251–12255.38. Mohamed MN, Watts HD, Guo J, Catchmark JM, Kubicki JD (2010) MP2, density

functional theory, and molecular mechanical calculations of C-H...pi and hydrogen

bond interactions in a cellulose-binding module-cellulose model system. Carbohydr

Res 345:1741–1751.39. Spiwok V, et al. (2005) Modelling of carbohydrate-aromatic interactions: ab initio

energetics and force field performance. J Comput Aided Mol Des 19:887–901.40. Pell G, et al. (2003) Importance of hydrophobic and polar residues in ligand binding in

the family 15 carbohydrate-binding module from Cellvibrio japonicus Xyn10C. Bio-

chemistry 42:9316–9323.41. Parsiegla G, Reverbel C, Tardif C, Driguez H, Haser R (2008) Structures of mutants of

cellulase Cel48F of Clostridium cellulolyticum in complex with long hemi-

thiocellooligosaccharides give rise to a new view of the substrate pathway during

processive action. J Mol Biol 375:499–510.42. Flint J, et al. (2004) Ligand-mediated dimerization of a carbohydrate-binding mole-

cule reveals a novel mechanism for protein-carbohydrate recognition. J Mol Biol 337:

417–426.43. Jervis EJ, Haynes CA, Kilburn DG (1997) Surface diffusion of cellulases and their iso-

lated binding domains on cellulose. J Biol Chem 272:24016–24023.44. Igarashi K, et al. (2009) High speed atomic force microscopy visualizes processive

movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose. J Biol

Chem 284:36186–36190.45. Cosgrove DJ (1999) Enzymes and other agents that enhance cell wall extensibility.

Ann Rev Plant Phys Plant Mol Biol 50:391–417.46. Brás JL, et al. (2011) Structural insights into a unique cellulase fold and mechanism of

cellulose hydrolysis. Proc Natl Acad Sci USA 108:5237–5242.47. Davies GJ, Tolley SP, Henrissat B, Hjort C, Schülein M (1995) Structures of oligosac-

charide-bound forms of the endoglucanase V from Humicola insolens at 1.9 A reso-

lution. Biochemistry 34:16210–16220.48. Wood TM (1988) Preparation of crystalline, amorphous, and dyed cellulase substrates.

Methods Enzymol 160:19–25.49. Raghothama S, et al. (2000) Solution structure of the CBM10 cellulose binding module

from Pseudomonas xylanase A. Biochemistry 39:978–984.50. Jamal S, Nurizzo D, Boraston AB, Davies GJ (2004) X-ray crystal structure of a non-

crystalline cellulose-specific carbohydrate-binding module: CBM28. J Mol Biol 339:

253–258.51. Boraston AB (2005) The interaction of carbohydrate-binding modules with insoluble

non-crystalline cellulose is enthalpically driven. Biochem J 385:479–484.52. Pettersen EF, et al. (2004) UCSF Chimera—A visualization system for exploratory re-

search and analysis. J Comput Chem 25:1605–1612.

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