Binding Specificity and Thermodynamics of Cellulose-Binding

Article pubs.acs.org/Biomac

Binding Specificity and Thermodynamics of Cellulose-Binding Modules from Trichoderma reesei Cel7A and Cel6A Jing Guo†,‡ and Jeffrey M. Catchmark*,†,‡,§ †

Intercollege Graduate Degree Program in Plant Biology, §Department of Agricultural and Biological Engineering, ‡Center for NanoCellulosics, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *

ABSTRACT: In this work, Family 1 cellulose binding modules CBMCel7A and CBMCel6A were heterologously expressed and purified from Escherichia coli, and the binding properties between these CBMs and cellulose substrates were studied. Cellulose nanowhiskers (CNWs, the crystalline portion of cellulose), microcrystalline cellulose Avicel PH101 (partially crystalline cellulose), and phosphoric acid swollen cellulose (PASC, amorphous cellulose) were used as representative models for cellulose to better understand the binding interactions between the CBMs and different regions of native cellulose. Isothermal titration calorimetry (ITC) was combined with adsorption-isotherm experiment to analyze the thermodynamics of CBM binding to various cellulose substrates. N2 adsorption and static light scattering (SLS) data were used to estimate the accessible surface area of cellulose which was then used for ITC data analysis. A new method of determining the cellulose molarity based on the available surface area for CBM binding was developed, which allows different cellulose substrates to be compared for binding experiments. The ITC results showed that the binding constant (Ka) to crystalline CNWs was ∼105 M−1 for CBMCel7A, while ∼106 M−1 for CBMCel6A, suggesting a higher binding affinity of CBMCel6A to CNWs. For Avicel, lower binding constants for both CBMs were observed, and weak bindings to PASC were characterized, suggesting that the binding between CBMCel7A,Cel6A and cellulose to some extent relates to the crystallinity of cellulose. Additionally, the binding reactions were driven by a favorable enthalpy change, offset partially by an unfavorable entropy change. It is suggested that CBMCel6A preferentially binds to the reducing end of cellulose chain, while CBMCel7A does not show such end binding specificities. Cello-oligosaccharides less than two glucose units did not bind with CBMs, and improved binding affinities were observed for cello-oligosaccharides with longer glucose units.

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INTRODUCTION Cellulose is the most abundant, renewable, and environmental friendly biopolymer on earth. It is important for the recycling of photosynthetically fixed carbon and an almost inexhaustible raw material that has been widely used for centuries. Diverse cellulolytic enzymes have evolved to tap the recalcitrance of cellulose, and cellulose binding modules (CBMs) are protein modules that exist in most cellulose hydrolyzing enzymes.1 It has been suggested that the CBMs improve the binding of catalytic modules on insoluble cellulose substrates and, thus, enhance the hydrolyzing activity of the whole cellulases by increasing the effective enzyme concentration at the substrate surface.2−8 It is proposed that the specific hydrolysis of a cellulase comes from its specific adsorption on the substrate, and it is the CBM that is responsible for such adsorption to the cellulose substrate. Depending on different binding specificities, CBMs can be classified into three types. Type A CBMs are characterized by a planar hydrophobic surface, which is comprised of conserved aromatic amino acids. This binding surface could selectively interact with crystalline polysaccharides.1,5,6,9−11 Type B CBMs have a cleft consisting of a central strip of hydrophobic residues © 2013 American Chemical Society

with polar hydrogen-bonding residues to accommodate single polysaccharide chains. These CBMs bind to soluble cellooligosaccharides in addition to amorphous cellulose, and the binding is enthalpically driven, in which direct hydrogen bonds play an important role.12 In general, association constants of Type A CBMs to crystalline cellulose are in the 106 M−1 range.1,12,13 The bindings for Type B CBMs to amorphous cellulose and cello-oligosaccharide ligands are ∼104 M−1.12 Thermodynamics of these interactions are typically driven by large favorable enthalpy change, which is partially offset by an unfavorable entropy change.12 Type C CBMs interact primarily with mono- or disaccharides.7 Among these CBMs, Family 1 CBMs of cellobiohydrolases Cel7A and Cel6A from the filamentous fungus Trichoderma reesei (also called Hypocrea jecorina), one of the most potent cellulase producers,14 have the simplest structure compared to other families (Table 1). It has been suggested that CBMCel7A and CBMCel6A preferably bind to the crystalline regions of Received: May 28, 2012 Revised: January 30, 2013 Published: March 18, 2013 1268

dx.doi.org/10.1021/bm300810t | Biomacromolecules 2013, 14, 1268−1277

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Table 1. Amino Acid Sequences for Family 1 CBMCel7A and CBMCel6Aa

a

organism

enzyme

amino acid sequence

T. reesei T. reesei

Cel7A Cel6A

TQSHY GQCGGI GY SGPTVCASGTTCQVLNPYYSQCL CSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCL

Note: aromatic amino acids that are particularly involved in binding to cellulose are highlighted.

that larger binding affinity between CBM and cellulose crystal complex are obtained from the (110) surface than the (100) and (010) ones. Beckham et al.17 showed that several residues on the CBM hydrophobic surface are responsible for forming hydrogen bonds to the cellulose surface (Y5, Q7, N29, and Y32), which are also responsible for the observed processivity length scale of the CBM−cellobiose unit. The simulation energy model for the interaction between CBMCel7A and cellulose surface established by Bu et al.31 also suggested that CBM could recognize the hydrolyzed cellulose chain ends and provide a driving force for the processive hydrolysis. When comparing the differences in binding ability of W and Y, Taylor et al. concluded that a 2-fold increase in binding affinity was obtained by mutating a tyrosine to tryptophan of Family 1 CBM.32 Taylor et al.32 also suggested that O-glycosylation near the CBM binding face affected Family 1 CBM binding affinity. In natural Cel7A, expressed in T. reesei, glycosylation might be present in the linker peptide and CBM, which may affect the binding of whole enzyme Cel7A onto cellulose.33 The CBMs in the present study are expressed in E. coli, thus, these CBMs are not glycosylated. To gain a better understanding of CBM binding to insoluble cellulose with different crystallinities, binding studies on CBMCel7A and CBMCel6A to insoluble cellulose and soluble cello-oligosaccharides were performed using ITC and adsorption-isotherm experiments, with the aim of determining the binding affinities and binding sites of these two CBMs on cellulose, as well as and the thermodynamic driving force(s) for binding. In addition, the end binding specificities of these CBMs toward cellulose reducing/nonreducing ends have also been characterized. Studying the mechanisms of CBM− cellulose interaction is fundamental in the process of understanding cellulose degradation.

cellulose and also demonstrate modest binding affinities to amorphous regions of cellulose and some cello-oligosaccharides.15 In addition, the NMR structure of Cel7A is well-known.9 CBMCel7A is comprised of irregular triple-stranded β-sheets with a flat surface formed by three aromatic amino acid residues (Y5, Y31, and Y32, numbering according to Kraulis et al.9), and these tyrosines are suggested to be involved in the hydrophobic interaction of the CBM with the cellulose substrate.1,16 Asparagine (N29) and glutamine (Q34) residues on the hydrophobic surface of the CBM are considered to serve as anchors to stabilize the interaction between the CBM and cellulose.17 The structure of CBMCel6A is very similar to CBMCel7A, and the amino acid residues essential for binding are highly conserved in various cellulose hydrolyzing enzymes among fungal species.18 It has been proposed that hydrogen bonding, van der Waals interaction (in particular, the CH-π stacking interaction between aromatic ring and glucose ring) contribute to the binding interaction between CBMs and carbohydrates.19−22 Although the binding between these CBMs to various cellulose substrates has been extensively studied (Table 2), Table 2. Summary of Reported Binding Affinities for Family 1 Cel7A, Cel6A, CBMCel7A, and CBMCel6A resource

method

Cel7A

T. reesei

Cel7A

T. reesei

CBMCel7A

T. reesei

CBMCel7A

T. reesei

CBMCel7A

T. reesei

Cel6A

T. reesei

CBMCel6A

T. reesei

binding isotherm binding isotherm binding isotherm binding isotherm binding isotherm binding isotherm binding isotherm

substrate

Ka (liters/g; pH 5.0, 4 °C)

ref

BC

5

5

BMCC

43 ± 2

23

BMCC

2.7 ± 0.2

23

BC

0.8

5

TC

1.7

1

BMCC

5.8 ± 0.2

23

BMCC

1.2 ± 0.2

23

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MATERIALS AND METHODS

Protein Expression and Purification. CBMCel7A and CBMCel6A were purified from E. coli, as described in detail in the Supporting Information. Cellulose Substrates Preparation. Microcrystalline cellulose (Avicel PH101) was purchased from FMC International. Avicel has been chosen as representative cellulose consisting of both crystalline and amorphous portions. Cellulose nanowhiskers (CNWs), which are the highly crystalline portions of the microfibril, were produced from Whatman CF11 cellulose by sulfuric acid (63.5%, w/w) hydrolysis according to the procedure implemented by Bondeson et al.34 A 55 g aliquot of cellulose was added into 1 L of 0.1 M NaOH solution with stirring for 1−2 h, followed by several washing steps to eliminate residual NaOH. The washed cellulose was then added into 500 mL of sulfuric acid (63.5%, w/w), the hydrolysis was carried out under 45 °C with continuous stirring for 90 min and further hydrolysis was stopped by additional washing with deionized (DI) water, after which the pH was neutralized by dialysis with DI water and CNW suspensions was obtained by sonication. Phosphoric acid swollen cellulose (PASC) is produced by phosphoric acid treatment of Avicel PH101, as reported previously.35 PASC is considered to be predominately amorphous cellulose. Glucose, cellobiose (Sigma-Aldrich), cellotriose, cellotetraose, cellopentaose, and cellohexaose were purchased from Seikagaku (Tokyo, Japan).

until now, the driving force(s) and mechanism(s) of CBMCel7A and CBMCel6A adsorption to cellulose are still not well understood due to the complicated structures of cellulose and cellulose−CBM complexes. More interestingly, it has been suggested that Cel7A and Cel6A act synergistically for cellulose hydrolysis such that treatment of crystalline cellulose with both Cel6A and Cel7A resulted in a significant increased concentration of mobile enzyme molecules on the cellulose surface and thus dramatically faster cellulose degradation than was the case with Cel7A alone.24 Cel7A preferentially releases cellobiose from the reducing end while Cel6A preferentially cleaves from the nonreducing end.25−29 However, it is still unknown that whether or not the end hydrolyzing selectivity arises from the binding specificity of these two CBMs. Several simulation studies have been done to explore the Family 1 CBMCel7A binding on the cellulose surface. Yui et al.30 showed 1269


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Reduced Cellulose Preparation. Reduced CNWs or Avicel were prepared following Boraston et al.’s method.36 Generally, 1 g of CNWs or Avicel PH101 was first suspended in 15 mL of 100 mM NH4OH (pH 10.8), after which 150 mg of NaBH4 was added. The mixture was incubated overnight with stirring at room temperature, after which the reduced CNWs or Avicel were obtained by centrifugation with several washing steps with DI water. The obtained cellulose was resuspended in 10 mL of DI water. The free reducing ends in the unmodified cellulose and reduced cellulose were quantified by BCA assay37,38 by using glucose as a standard. Cellulose Substrate Characterization. 1. Available Binding Surface Area on Cellulose. Cellulose has a heterogeneous and porous structure so that the possible binding surfaces for CBMs are limited. The available binding surface accessible to CBMs on cellulose was estimated by N2 adsorption method based on Brunauer−Emmett− Teller (BET) calculation,39,40 which was used for ITC data analysis. 2. Molecular Weight Determination of Cellulose. The molecular weight of cellulose was determined by static light scattering (SLS) technique, and this mass molarity was also used for ITC data analysis. The measurements were performed in a Zetasizer Nano ZS instrument ZEN3500 (Malvern Instrument) at room temperature. A series of cellulose suspensions dialyzed against Millipore DI water were first sonicated and then diluted with DI water to obtain concentrations in the range of 0.125 to 2.0 mg/mL. The wavelength of the incident laser light was λ = 633 nm. The cellulose samples were loaded in a square glass cuvette. The cuvette was rinsed/cleaned with filtered DI water before use. After calibration with filtered toluene to establish the reference scattering intensity, a number of prepared cellulose concentrations were measured. The data were evaluated and recorded in the form of Debye plots. CBM−Cellulose Binding Affinity Characterization. 1. Binding Affinities Determined by Adsorption-Isotherm Experiments. Binding constants of CBMs and insoluble cellulose at 25 °C were determined from adsorption-isotherms, as described previously. 13,41,42 All adsorption-isotherm measurements were carried out in 1.5 mL Eppendorf tubes containing serial dilutions of protein CBMs mixed with an equal volume of an aqueous suspension of CNWs to a final concentration of 1 mg/mL in 50 mM phosphate buffer (pH 5.5). All adsorption experiments were in triplicate. Control tubes contained CBMs only (no CNWs). Each solution was vortexed and then rotated end-over-end for 16 h to allow the adsorption system to equilibrate. The samples were then centrifuged at 25 °C and 10000 rpm for 10 min to remove the protein-bound CNWs. The clear supernatant with unbound CBMs was collected and CBM protein concentration was measured. The equilibrium association constants (K a ) were determined by nonlinear regression of bound versus free protein concentrations to Langmuir model as described previously.13 Two models for equilibrium binding were compared by nonlinear regression of the data to a Langmuir type one (eq 4) and type two (eq Eq. 5) binding site(s). For a reversible ligand−macromolecule interaction, the equilibrium can be expressed in the following terms:

where [N0] is the concentration of the total available binding sites in the absence of ligand. The combination of eqs 2 and 3 yields the following equation,

[B] =

Kd

[B] =

[B] [N ][F ]

(Eq. 5)

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RESULTS Cellulose Accessibility to CBMs Determined by Two Methods. 1. Available Surface Area for CBM Binding Based on Surface Area Obtained by N2 Adsorption. Available surface area of cellulose was quantified by N2 adsorption method. The total surface area was calculated by BET method from the linear part of the adsorption isotherm, at pressures 0.05 < P/P0 < 0.30, and by the BJH method where the specific surface area allowing for CBM binding is determined as a function of pore size.40 However, the cellulose surface varies based on the crystal structure, which depends on the origin and processing conditions and may also vary throughout the sample. It has been suggested that although Family 1 fungal CBMs exhibit a preference for the hydrophobic face, they may be capable of binding all regions of the cellulose surface.42 For simplicity, in this analysis we assume that all faces of the cellulose surface are accessible for CBM binding. However, in addition to variations in surface chemistry, cellulose also exhibits a variation in porosity. To address this intricacy in subsequent calculations,

(1)

(2)

where [N] is the concentration of available binding sites on cellulose (moles·g cellulose−1),

[N ] = [N0] − [B]

[N1]K a1[F ] [N2]K a2[F ] + 1 + K a1[F ] 1 + K a2[F ]

where [N1] and [N2] are the density of binding sites on the cellulose, 1, 2 refers to binding site 1 and binding site 2. Ka1 and Ka2 represent the binding constants for binding sites 1 and 2. 2. Binding Constants Determined by Isothermal Titration Calorimetry (ITC). ITC was performed using a Nano 2G ITC (TA Instruments). Briefly, all samples were dissolved in the same buffer containing 50 mM phosphate buffer (pH 5.5). All solutions were thoroughly degassed under vacuum prior to measurements. The system was allowed to equilibrate for 30 min at 25 °C with stirring at 400 rpm to ensure rapid mixing and equilibrium. Titrations were performed by injecting CBM protein solution from the syringe into the ITC sample cell containing cellulose substrate suspensions with 220 s between each injection. The concentrations of CBMCel7A and CBMCel6A were determined spectrophotometrically at 280 nm. C values (C = Ka × M × n, where M is the macromolecule concentration, and n is the number of binding sites on the macromolecule) are always adjusted to be between 10 and 100. A blank experiment to evaluate the heat of dilution contributed from peptide dilution was performed at 25 °C using identical injections of peptide solution into buffer, and all ITC data were corrected for the heat of dilution by subtracting the blank from test experiment. At least three independent titration experiments were performed to determine the binding constant of CBM to cellulose. A thermodynamic profile of binding interaction was determined by fitting the data to an independent binding site interaction model. ITC directly measures binding constant (Ka) and enthalpy change (ΔH), from these parameters and the equations ΔG = −RT ln(Ka) and ΔG = ΔH − TΔS, free Gibbs energy change ΔG and entropy change ΔS values can be calculated.

At equilibrium, an isotherm of [B] versus [F] was generated, [N] is the concentration of available binding sites, [F] is the concentration of free CBM (moles·g cellulose−1), which was determined from the absorbance at 280 nm using the extinction coefficient of the purified CBMs; the concentration of bound protein, [B], was determined from the difference between the initial protein concentration and [F]; and the equilibrium association constant (also called binding constant or adsorption constant, Ka, L·mol−1) was determined as following: Ka =

(4)

The one binding site Langmuir isotherm fit is widely used because in most cases it provides a good fit to the data. Besides, this one site fit represents a simple mechanistic model to describe the binding kinetic properties of various cellulase−cellulose systems. However, the single binding site model is not applicable when there are multiple types of adsorptions sites.43,44 In the case of multiple binding sites, alternative equilibrium model to simple Langmuir adsorption (two-site adsorption models45,46) can be used. In model II, two independent classes of binding sites are characterized by

Ka

N+F⇄B

[N0]K a[F ] 1 + K a[F ]

(3) 1270


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we use the surface area accessible to CBMs, that is, the surface area associated with porosities larger than the largest approximate size of the CBMs. For CBMCel7A, the NMR structure is known and the approximate dimensions are 3.0 × 1.8 × 1.0 nm3.9 The crystal structure of CBMCel6A is predicted to be of comparable size to CBMCel7A, and thus, the smallest pore size for CBMs is assumed to be 3 nm. Table 3 summarizes the total surface area (ABET) and specific surface area (SSA) associated with pore sizes (D) larger than 3 nm.

c=

where NA is Avogadro’s constant (6.023 × 1023 molecule/mol). Similarly, the total number of available unit surface area (Au) of Avicel and PASC can be calculated, and consequently, the molar concentration of Au on Avicel and PASC was obtained as 0.0056 and 0.0934 mM, respectively. 2. Cellulose Accessibility Based on Cellulose Molecular Weight (Mw). The molecular weight of CNWs was determined by SLS, in which the light scattering of CNWs at different concentrations were measured and applied with the Rayleigh equation: KC/Rθ = ((1/Mw) + 2A2C)P(θ), in which R θ is the Rayleigh ratio of scattered light to incident light of the CNWs; Mw is the molecular weight of CNWs; A2 is the second virial coefficient; C is the concentration of CNW suspension; P(θ) is the angular dependence of the sample scattering intensity, and K is the optical constant, given by K = (2π2/(λο4NA))(nο(dn/ dc))2, where λo is the laser wavelength; no is the solvent refractive index, and (dn/dc) is the differential refractive index increment. This is the change in refractive index as a function of the change in concentration.47 Toluene was initially measured as a standard pure liquid with a known Rayleigh ratio, and then the Mw was determined based on the scattering intensity of the CNWs relative to that of toluene. As shown in equation Rθ = ((IAnο2)/(ITnT2))RT, in which IA is the residual scattering intensity of the CNWs. IT is the toluene scattering intensity, nT is the toluene refractive index, and RT is the Rayleigh ratio of toluene. The intensity of scattered light that a CNW suspension produces is proportional to the product of the weight-average molecular weight and the concentration of the particle. The SLS measures the intensity of scattered light (K/CRθ) of various concentrations (C) of sample at one angle. This is compared with the scattering produced from a standard (i.e., toluene). The Mw of CNWs was determined from the intercept on the x-axis and shown in Figure 1 (CNWs were considered to have a rod-like shape in the measurement). It is shown that the Mw of CNWs is 778 kDa. Because the concentration of CNWs is C = 4.2 g/L, the molar concentration of CNWs is c = (C/Mw) = ((4.2 g/L)/(7.78 × 105 g/mol)) = 0.0054 mM. However, in the case of PASC, SLS cannot be used for the Mw calculation because there was no even suspension formed. The calculated molarities of CNWs

Table 3. Specific Surface Area of Cellulose Substrates for CBM Binding by BJH Calculation Using N2 Adsorptiona

a

sample

BET surface (>0.4 nm) ABET (m2/g)

SSA for CBMCel7A/Cel6A (D > 3 nm)

Avicel CNWs PASC

2.4 ± 0.7 5.1 ± 0.7 16.3 ± 1.0

0.8 ± 0.0 4.4 ± 0.4 13.4 ± 0.2

SSA, specific surface area; D, diameter.

To calculate the binding constant associated with the CBMs, the molarity of the cellulose substrates needs to be quantified. This represents a key issue in the study of materials that bind cellulose, as many different cellulose forms exist, making a comparison between binding studies using different forms of cellulose difficult. This issue has not been addressed in the literature, where typically the approximate cellulose molecular weight is used. This method, however, does not accurately represent the available binding area. In this work, we assume a unit cellulose surface area Au, where Au = 1.0 × 1.0 nm2. For example, in the case of CNWs, the number of unit area sites available for binding is then given by Nu = =

Nu 1.848 × 1019 molecule/L = = 0.0306 mM NAV 6.023 × 1023 molecule/mol

SSA × C Au 4.4 m 2/g × 4.2 g/L 1.0 × 10−18 m 2

= 1.848 × 1019 A u /L

where SSA [SSA = 4.4 (±0.4) m2/g (Table 3)] is the available area for CBM binding, which exhibits a pore size greater than, in this case, 3 nm. The concentration of CNWs (from cotton CF11) is C = 4.2 g/L. Therefore, the molar concentration of available unit surface area sites of CNWs is calculated as

Figure 1. Weight-average molecular weight (Mw) of CNW suspension determined by SLS. 1271


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Figure 2. Adsorption-isotherms of CBMCel7A,Cel6A binding to CNWs, Avicel, and PASC in 50 mM phosphate buffer at pH 5.5. Ka were determined by nonlinear regression of [B] vs [F] to Langmuir one binding site model and two binding sites model for binding to CNWs and Avicel. Only one binding site model was fit for PASC because very weak bindings were observed.

ascending binding isotherms for CBMs on CNWs, Avicel, and PASC in 50 mM phosphate buffer at pH 5.5 and 25 °C. The concentration of purified CBMCel7A, Cel6A peptides were determined by UV absorbance at 280 nm using a calculated molar extinction coefficient of 5960 M−1 cm−1 and 15470 M−1 cm−1 for CBMCel7A and CBMCel6A, respectively.48 Two putative models for equilibrium binding were compared to determine the binding constants for binding of CBMs to insoluble cellulose substrates with varied crystallinities. As a widely accepted model, the Langmuir equation represents a

based on these two methods are not exactly comparable since different definitions of molarity were adopted in each calculation. The former is the molar concentration of specific surface area on CNWs for CBM binding, while the latter is the molar concentration of the mass of CNWs. CBM−Cellulose Substrate Binding Characterization. Adsorption-Isotherm Experiment. The cellulose adsorptionisotherm is a method adapted to the direct measurement of the partitioning of CBMs between the bound and free status when using cellulose as an insoluble sorbent. Figure 2 shows the 1272


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simple mechanistic model and always provides good fit.13 Figure 2a−f show by fitting the CBMs adsorption on the cellulose substrates using the Langmuir isotherm equation, Eb = (BmaxEf/(Kd + Ef)) (Eb is bound CBMs, μmol/g cellulose; Ef is free CBMs, μM; Bmax is the maximum CBM adsorption/g cellulose; Kd is dissociation constant = 1/Ka), assuming that adsorption could be described by a single adsorption equilibrium constant and a specific adsorption capacity. The maximum CBMCel7A adsorption on CNWs is 5.50 μmol/g CNWs. Using the approximate dimensions of 30 × 18 Å (=540 Å2) of CBMCel7A by NMR,9 the minimum covered area on cellulose by CBMCel7A can be calculated as

highest crystalline contents. The Ka values for Avicel, which is partially crystalline cellulose, are 2.8 × 104 M−1 and 2.45 × 104 M−1. However, CBMs have the lowest affinity for amorphous cellulose with K a of ∼10 3 M −1 . These CBMs have approximately 100-fold greater affinity for highly crystalline cellulose than for amorphous cellulose, and Ka for adsorption of CBMs to cellulose decreases with the decreased crystalline contents in cellulose substrates. This observation may suggest that the strong affinity of CBMs for CNWs probably relate to the highly ordered cellulose structure. Thermodynamics of CBM-Cellulose Binding Characterized by ITC. The thermodynamics of CBMCel7A and CBMCel6A binding with cellulose were investigated by ITC. Titrations of the CBMs into CNWs yielded negative power deflections, indicating exothermic binding and integrated isotherms of sigmoid shape. These calorimetric isotherms also appear to show one class of binding site on CNWs for both CBMs. The molarities of CNWs calculated by both accessible surface area and SLS methods were applied for ITC data analysis, and it appeared the resulting thermodynamic data were comparable for these two methods. Similarly, the molarities of Avicel calculated by two methods were applied for ITC analysis. In this study, the binding constants were calculated by both Langmuir isotherm and ITC measurement. The Langmuir isotherm is a most common method for measuring CBM adsorption, which provides a simple mechanistic model for the binding affinities of various cellulase−cellulose systems. However, some limitation of this method should not be ignored, which was discussed in Zhang and Lynd’s paper.50 The binding constants generated by ITC are considered to be more reliable because ITC is an improved method compared to Langmuir fitting. The binding constant of CBMCel7A to CNWs was on the order of 105 M−1, and that to Avicel was around 104 M−1 (Table 5), suggesting a relatively high binding affinity toward crystalline CNWs. The negative Gibbs free energy indicated that this binding reaction was spontaneous. In addition, these data demonstrated that dominating favorable changes in enthalpy were partly compensated for by unfavorable changes in the entropy. It has been reported that the enthalpic driving force mainly comes from the forming and breaking of hydrogen bonds and van de Waals interactions, and so on.12 The observed results imply that binding is enthalpically driven and consistent with a structural model17 involving hydrogen bonding between the equatorial hydroxyls of the glucose rings and polar amino acid chains. It is possible that hydrogen bonds might be formed between the hydroxyl groups of the cellulose and tyrosines Y5, Y31, Y32, glutamine Q7, Q34, and asparagine N29. This is consistent with Beckham et al.’s simulation that hydrogen bonding interactions are highly likely dominate the CBM processivity on the hydrophobic surface of cellulose.17 Thermodynamic characterization of the interactions can help to elucidate the nature of the forces driving the binding reaction in solution and this information can be used for engineering biologically based linker molecules which integrate CBMs. Due to the sequence homology between CBMCel7A and CBMCel6A (Table 1), two CBMs share similarities in binding properties toward cellulose. It is shown from the thermodynamic data that CBMCel6A showed similar binding property to that of CBMCel7A. However, the binding constant of CBMCel6A is even larger than CBMCel7A, which may be due to the different amino acid composition of each protein module. It is proposed

S = Bmax × NA × SCBMCel7A = 5.50 μmol/g × 6.02 × 1023 molecules/mol × 30 × 18 × 10−20 m 2 = 17.9 m 2 × g

in which S represents the surface area of cellulose accessible to CBMCel7A and NA = 6.023 × 1023 molecules/mol. Moreover, the cellobiose lattices occupied CBMCel7A are calculated as follows: α = =

SCBMCel7A SG2 30 × 18 × 10−20 m 2 = 9.8 5.512 × 10−19 m 2

where SG2 = the area of cellobiose lattice49 (5.512 × 10−19 m2). It suggests that CBMCel7A can occupy 9.8 cellobiose lattices when adsorbed onto CNWs, which is in great agreement with previous hypothesis that CBMCel7A might occupy about 10 cellobiose lattices based on nuclear magnetic resonance data.5,9 Alternatively, two independent site adsorption binding model was also applied assuming the heterogeneity in cellulose surface which may impart multiple types of adsorption sites, as well as the possible interaction among the adsorbing CBM proteins (Figure 2g−j). In this study, both one-site binding and two-site binding model were used to fit the data. It is shown that the binding constants calculated by two-site binding model are similar to that of one-site binding model (on the same order). It appears that no extra benefit is provided by two-site binding model compared to one-site binding model. Therefore, one site binding model was used for our analysis. From Table 4 it seems that the adsorption of CBMs to cellulose is greatly influenced by the cellulose structure. The affinities are varied with respect to the different crystallinities of the cellulose tested. CBMs have the highest binding affinity for CNWs (1.9 × 105 M−1, 2.7 × 105 M−1), which contain the Table 4. Affinity Constants of CBMCel7A,Cel6A Binding to Insoluble Cellulose Determined by Adsorption-Isothermsa CBMCel7A

CBMCel6A

substrate

Ka

(×10−5 M−1)

Bmax (μmol/g)

Ka

(×10−5 M−1)

Bmax (μmol/g)

CNWs Avicel PASC

1.9 0.28 0.0135

5.49 4.82 21.01

2.7 0.245 0.0347

15.28 19.97 17.5

a

CNWs, cellulose nanowhiskers; PASC, phosphoric acid swollen cellulose. 1273


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Table 5. Thermodynamics of CBMCel7A,Cel6A Binding to Cellulose Determined by ITC at 25 °Ca Ka (M−1)

CBM CNWs Avicel PASC modified CNWs modified Avicel

CBMCel7A CBMCel6A CBMCel7A CBMCel6A CBMCel7A CBMCel6A CBMCel7A CBMCel6A CBMCel7A CBMCel6A

(3.31 (1.18 (5.34 (1.23 NA NA (5.13 (8.40 (5.37 (6.92

ΔH (kJ/mol)

± ± ± ±

0.43) 0.54) 0.36) 0.22)

× × × ×

105 106 104 105

± ± ± ±

1.30) 0.20) 0.96) 1.26)

× × × ×

104 104 104 104

−76.31 ± 3.76 −145.10 ± 6.44 −17.02 ± 13.82 −24.44 ± 4.93 NA NA −55.57 ± 2.50 −40.05 ± 0.52 −44.01 ± 9.92 −38.14 ± 0.19

ΔG (kJ/mol)

TΔS (kJ/mol)

−31.58 −34.19 −26.96 −29.01 NA NA −26.82 −11.97 −26.96 −27.59

−43.53 ± 1.60 −111.26 ± 7.16 9.95 ± 5.93 4.57 ± 4.73 NA NA −28.75 ± 1.98 −28.08 ± 0.06 −17.06 ± 10.03 −10.54 ± 0.65

± ± ± ±

0.32 1.16 0.20 0.38

± ± ± ±

0.66 0.57 0.46 0.46

a

NA represents no binding experiment was performed using ITC. This is due to the heterogeneity of PASC; no even suspension could be used for titration. Errors represent the standard deviations of quadruplicate experiments. Modified CNWs/Avicel represents cellulose sample with decreased number of reducing ends, which is obtained by NaBH4 modification.

Figure 3. The proposed mode of operation of the exoglucanases Cel7A and Cel6A from T. reesei.

105 M−1, less than one order of difference in binding affinity is observed. It is of interest to notice that the binding of CBMCel6A to a great extent depends on the existence of reducing ends in cellulose, suggesting that the recognition site for CBMCel6A is the reducing end of cellulose. In contrast, for CBMCel7A only minor difference is observed when binding to modified cellulose compared to unmodified cellulose, and this difference may result from the sheltering effect of the possible binding sites along the glucan chain by NaBH4. This result indicates that CBMCel7A does not have binding specificity to the reducing ends. CBMCel6A binds to unmodified Avicel with a binding constant of 1.23 (±0.22) × 105 M−1 with reducing ends concentration of 21.3 nmol/mg cellulose. When binding to the NaBH4 treated Avicel, which has 11.7 nmol/mg of reducing ends, the binding constant is reduced to 6.92 (±1.26) × 104 M−1. It appears that NaBH4 treatment of Avicel affects the binding affinity of CBMCel6A. An almost 50% binding affinity loss, along with 50% reduction of reducing ends in Avicel, suggests it is highly likely that CBMCel6A has ability to recognize and bind to the reducing ends of glucose chains. CBMCel7A binds to unmodified Avicel with a binding constant of 5.34 (±0.36) × 104 M−1, but also exhibits a very similar binding constant of 5.37 (±0.96) × 104 M−1 to NaBH4-treated Avicel. The comparable binding constants suggest that none or very few of the reducing ends of glucan chains act as binding sites for CBMCel7A. The previous theories25−29 only discussed about the binding specificities of the whole cellulase enzyme (Cel7A and Cel6A) instead of the binding specificities of the binding domains. The binding specificity of CBMs is proposed and shown in Figure 3. The catalytic domain of CBMCel7A (large green dot) hydrolyzes cellulose from the reducing ends, when the whole enzyme is aligned with the cellulose chain, the binding domain (small green triangle) may recognize and bind to the nonreducing end other than the reducing end, and thus help to accommodate catalytic module to the reducing ends. Similarly, Cel6A hydrolyzes cellulose chain from the non-

that a hydrophobic surface formed by a YYY (Y5, Y31, and Y32) composition of CBMCel7A is the binding surface for crystalline cellulose.1,9,51 In CBMCel6A, instead of a YYY composition, the hydrophobic surface is composted of a WYY composition (W5, Y31, and Y32). Lower binding constant is observed with YYY in CBMCel7A compared to WYY in CBMCel6A. The binding between CBMCel6A and CNWs, on the other hand, showed slightly larger enthalpy and entropy change, probably arising from better hydrogen bonding and/or CH-π stacking interactions and there were more water molecules displaced from the surface of both CBMCel6A and CNWs when binding occurred. CBM Reducing/Nonreducing End Binding Specificity. Previous studies suggested that the whole enzyme Cel7A and Cel6A had specificities in hydrolyzing cellulose chains. It was discovered that Cel7A would hydrolyze cellulose from the reducing ends while Cel6A would digest from the nonreducing ends.27,28 However, it is still unclear whether such enzyme hydrolyzing specificity results from the binding specificity of its CBM. In order to identify the glucan chain end binding specificities of these two CBMs, the reducing ends of cellulose were modified by NaBH4 to reduce the hemiacetals at C-1 and, thus, to open the glucose ring.36,52,53 Unmodified CNWs have a concentration of reducing ends of 43.4 nmol/mg cellulose (quantified by BCA assay). When treated with NaBH4, the concentration of reducing ends decreases to 21.4 nmol/mg cellulose, thus, approximately 50% of the reducing ends disappear after NaBH4 modification. Coincidentally, a binding constant of 8.4 (±0.2) × 104 M−1 is observed between CBMCel6A and modified CNWs compared to 1.18 (±0.54) × 106 M−1 with unmodified CNWs. An almost two-order decrease of the binding affinity implies that the reducing ends appear to contain the recognition determinants required for their binding to CBMCel6A. On the other hand, it is observed that CBMCel7A binds to modified CNWs with binding affinity constant of 5.13 (±1.30) × 104 M−1. Compared to its binding to unmodified CNWs, with binding affinity of 3.31 (±0.43) × 1274


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CNWs than that of CBMCel7A, having a YYY (Y5, Y31, Y32) configuration in the putative binding motif. This is the first time that the molar concentration of cellulose substrates was accurately addressed for ITC data analysis. The molar concentration was quantified by both accessible unit surface area based on the surface area by N2 adsorption and weight-average molecular weight of cellulose suspension by SLS. This is important for comparing ITC data for the binding of peptides or other molecules to different forms of cellulose. Our result shows that the accessible surface area of cellulose to CBM predicted from adsorption-isotherm is larger than the surface area measured by N2 adsorption. This may be caused in part by the overestimation of the maximum loading of CBMs on cellulose due to protein−protein interaction of these CBMs or packing behavior of the CBMs bound to cellulose. On the other hand, this might also imply that more surface area is created in the binding process, which is supported by previous studies that some CBMs may appear to disrupt the noncovalent interactions between glucan chains from the crystalline cellulose surface.11,54 In particular, Lehtio et al.55 claimed that both CBM1s (CBMCel7A and CBMCel6A) and CBM3a bound to the 110 face (cellulose Iα, Valonia cellulose), and these hydrophobic corners were usually disrupted by CBM binding, giving rise to larger surface area. These results suggest that in our study, when the outer layer of the cellulose crystal obtuse corner is disrupted by CBM, more internal 200 faces (200 face in the case of cellulose Iß, 110 face in the case of cellulose Iα) are exposed and CBMs could consequently bind onto the newly exposed 200 surface. This additional surface area may not be able to be measured by N2 adsorption. Based on the proposed 3D structure of plant cellulose crystals of Iβ,56 optimum CH-π stacking interaction between aromatic ring and glucose ring is possibly obtained only on the obtuse corners of the crystal with exposed areas on the 200 surface.22 Hydrogen bonding can be formed between 200, 110, 1¯10 surfaces and CBMs because the three hydroxyl groups (O2, O3, and O6) are available to form hydrogen bonds in each cellobiose unit. Additionally, because the area of exposed 200 surface is small, van der Waals interaction may be correspondingly small, making it unable to stabilize the binding between CBM and cellulose. Quantification of the thermodynamic parameters reveals the physical process involved in the binding reaction. It has been reported that CBMCel7A and CBMCel6A bind to crystalline cellulose with a hydrophobic binding surface comprised by a number of conserved residues, including three aromatic amino acids that have been implicated in the binding reaction.1,16 In this study, the thermodynamic data characterized by ITC shows binding of CBMs to CNWs is exothermic. This exothermic binding probably results from favorable intermolecular hydrogen bond formation between the cellulose and the CBMs, formation of new water−water hydrogen bonds in the bulk solution, and formation of close van der Waals contacts between the cellulose and the CBMs. This is suggested by previous studies that favorable changes in enthalpy were very common in CBM−carbohydrate interactions 12,36,57 and resulted primarily from polar interactions, such as hydrogen bonding and van der Waals interactions. In this model, it is highly likely that CBMCel7A and CBMCel6A make enthalpic contacts with the largely inflexible CNW surface primarily due to an increased number of hydrogen bonds with optimal donor−acceptor geometry and distance at the CBM−cellulose interface, and to more favorable van der Waals interactions

reducing end, while the binding module (CBMCel6A) is likely binding to the reducing end. This finding is consistent with Igarashi et al.24 that the CBMCel6A binds very strongly to reducing ends, perhaps this is how Cel6A cleans up the socalled “traffic jams” that Igarashi and co-workers report when Cel7A enzymes get stuck. CBM Binding to Cello-Oligosaccharides. The binding affinities of CBMs to cello-oligosaccharides were also investigated using ITC (Figure 4). Results show that CBMCel6A

Figure 4. Binding constants (Ka) of CBMCel6A binding to cellulose and cello-oligosaccharides determined by using ITC at 25 °C. CBMCel7A shows similar binding characteristics toward cello-oligosaccharides.

binds to cellohexaose and cellopentaose and weakly to cellotetraose and cellotriose. There is no detectable binding to cellobiose or glucose, suggesting that cellotriose might be the smallest binding motif over which the CBM protein spans. Moreover, decreased binding affinities were observed along with a decreased length of cello-oligosaccharides.

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CONCLUSIONS AND DISCUSSION The binding of the Family 1 CBMs from T. reesei (CBMCel7A, CBMCel6A) to insoluble cellulose and soluble cello-oligosaccharides were characterized by both adsorption-isotherm experiment and ITC. These two CBMs bind both insoluble cellulose and some soluble cello-oligosaccharides with certain affinities. The strongest binding was observed between CBMCel7A/Cel6A and highly crystallized CNWs. A modest binding exists in Avicel (partially crystalline cellulose), while the binding to amorphous cellulose PASC is the weakest. This observation is consistent with the nature of Family 1 CBMs, which preferably bind to crystalline cellulose.45 Although, in nature, CBMs with cellulose binding activity are often found on insoluble crystalline cellulose, especially in plant cell walls, the relatively weak binding for cello-oligosaccharides reveals to some extent the structural binding mechanism between the CBMs and the substrate, demonstrating that three glucose units are probably the basic binding motif that CBMs can recognize and interact with. In addition, the affinity of CBMs for cellooligosaccharides may allow their application as purification tags. Several papers have shown that the affinity between tryptophan and glucose ring is higher than that between tyrosine and glucose unit,1 which might be the explanation why the Ka is higher for the binding of CBMCel6A, which has a WYY (W5, Y31, Y32) configuration in the putative binding motif, to 1275


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(3) Knowles, J.; Lehtovaara, P.; Teeri, T. Trends Biotechnol. 1987, 5, 255−261. (4) Din, N.; Gilkes, N. R.; Tekant, B.; Miller, R. C.; Warren, R. A.; Kilburn, D. G. Bio/Technology 1991, 9, 1096−1099. (5) Reinikainen, T.; Teleman, O.; Teeri, T. T. Proteins 1995, 22, 392−403. (6) Tormo, J.; Lamed, R.; Chirino, A. J.; Morag, E.; Bayer, E. A.; Shoham, Y.; Steitz, T. A. EMBO J. 1996, 15, 5739−5751. (7) Boraston, A. B.; Bolam, D. N.; Gilbert, H. J.; Davies, G. J. Biochem. J. 2004, 382, 769−782. (8) Hall, M.; Bansal, P.; Lee, J. H.; Realff, M. J.; Bommarius, A. S. Bioresour. Technol. 2011, 102 (3), 2910−2915. (9) Kraulis, P. J.; Clore, G. M.; Nilges, M.; Jones, T. A.; Pettersson, G.; Knowles, J.; Gronenborn, A. M. Biochemistry 1989, 28, 7241− 7257. (10) Xu, G. Y.; Ong, E.; Gilkes, N. R.; Kilburn, D. G.; Muhandiram, D. R.; Harris-Brandts, M.; Carver, J. P.; Kay, L. E.; Harvey, T. S. Biochemistry 1995, 34, 6993−7009. (11) Din, N.; Forsythe, I. J.; Burtnick, L. J.; Gilkes, N. R.; Miller, R. C., Jr.; Warren, R. A. J.; Kilburn, D. G. Mol. Microbiol. 1994, 11, 747− 755. (12) Tomme, P.; Creagh, A. L.; Kilburn, D. G.; Haynes, C. A. Biochemistry 1996, 35, 13885−13894. (13) Gilkes, N. R.; Jervis, E.; Henrissat, B.; Tekant, B.; Miller, R. C.; Warren, R. A.; Kilburn, D. G. Chem. Biol. Chem. 1992, 267, 6743− 6749. (14) Ståhlberg, J.; Johansson, G.; Pettersson, G. Eur. J. Biochem. 1988, 173 (1), 179−183. (15) Nieves, R. A.; Ellis, R. P.; Todd, R. J.; Johnson, T. J. A.; Grohmann, K.; Himmel, M. E. Appl. Environ. Microbiol. 1991, 57, 3136−3170. (16) Gilkes, N. R.; Henrissat, B.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J. Microbiol. Rev. 1991, 55, 303−315. (17) Beckham, G. T.; Matthew, J. F.; Bomble, Y. J.; Bu, L.; Adney, W. S.; Himmel, M. E.; Nimlos, M. R.; Crowley, M. F. J. Phys. Chem. B 2010, 114 (3), 1447−1453. (18) Takashima, S.; Ohno, M.; Hidaka, M.; Nakamura, A.; Masaki, H.; Uozumi, T. FEBS Lett. 2007, 581 (30), 5891−5896. (19) Quiocho, F. A. Annu. Rev. Biochem. 1986, 55, 287−315. (20) Quiocho, F. A. Biochem. Soc. Trans. 1993, 21, 442−448. (21) Vyas, N. K. Curr. Opin. Struct. Biol. 1991, 1, 732−740. (22) Mohamed, N. A. M.; Watts, H. D.; Guo, J.; Catchmark, J. M.; Kubicki, J. D. Carbohydr. Res. 2010, 345 (12), 1741−1751. (23) Palonen, H.; Tenkanen, M.; Linder, M. Appl. Environ. Microbiol. 1999, 65 (12), 5229−5233. (24) Igarashi, K.; Uchihashi, T.; Koivula, A.; Wada, M.; Kimura, S.; Okamoto, T.; Penttilä, M.; Ando, T.; Samejima, M. Science 2011, 333 (6047), 1279−1282. (25) Chanzy, H.; Henrissat, B. FEBS Lett. 1985, 184 (2), 285−288. (26) Claeyssens, M.; Tomme, P.; Brewer, C. F.; Hehre, E. J. FEBS Lett. 1990, 263 (1), 89−92. (27) Biely, P.; Vrsanska, M.; Claeyssens, M. TRICEL93 Symposium on Trichoderma reesei Cellulases and Other Hydrolases. In Foundation for Biotechnical and Industrial Fermentation Research; Suominen, P., Reinikainen, T., Eds.; University of WisconsinMadison: Madison, Wisconsin, 1993; pp 99−108. (28) Barr, B. K.; Hsieh, Y. L.; Ganem, B.; Wilson, D. B. Biochemistry 1996, 35, 586−592. (29) Teeri, T. T.; Koivula, A.; Linder, M.; Wohlfahrt, G.; Divne, C.; Jones, T. A. Biochem. Soc. Trans. 1998, 26, 173−177. (30) Yui, T.; Shiiba, H.; Tsutsumi, Y.; Hayashi, S.; Miyata, T.; Hirata, F. J. Phys. Chem. B 2010, 114 (1), 49−58. (31) Bu, L.; Beckham, G. T.; Crowley, M. F.; Chang, C. H.; Matthews, J. F.; Bomble, Y. J.; Adney, W. S.; Himmel, M. E.; Nimlos, M. R. J. Phys. Chem. B 2009, 113 (31), 10994−11002. (32) Taylor, C. B.; Talib, M. F.; McCabe, C.; Bu, L.; Adney, W. S.; Himmel, M. E.; Crowley, M. F.; Beckham, G. T. J. Biol. Chem. 2012, 287, 3147−3155.

between the aromatic ring hydroxyl groups and the cellulose surface. Unfavorable changes in entropy which are observed in this study are also characteristic of carbohydrate-binding events and are thought to result mainly from the freezing of sugar and amino acid side chain conformations.12,58 Binding specificity is likely determined by the precise pairing of hydrogen bond donors and acceptors, electrostatic interactions, and so on, on the contacting CBM and cellulose surface. This study shows that both CBMCel7A and CBMCel6A preferentially bind to crystalline cellulose, which is consistent with the previous report that a Family I CBM prefers to bind to the crystalline regions of cellulose.45 The thermodynamic data presented in this study suggest that the binding preference to crystalline regions of cellulose may rise from the favorable enthalpic interactions (hydrogen bonding and van der Waals interactions) between CBMCel7A/Cel6A and crystalline cellulose. On the other hand, cellulase hydrolyzing specificities were reported in previous studies that Cel7A preferentially releases cellobiose from the reducing end while Cel6A preferentially cleaves from the nonreducing end.25−29 It is still unclear whether or not the end hydrolyzing selectivity arises from the binding specificity from these two CBMs. It is showed in this study that CBMCel7A and CBMCel6A may have different cellulose chain end binding specificities, suggesting the presence of cellulose chains with physical presentations that are distinguishable by the CBMs. Besides the binding to insoluble cellulose, these CBMs also show binding affinities to certain cellooligosaccharides, with an affinity approximately 1−2-orders of magnitude lower than their affinity for insoluble cellulose, implying that binding to insoluble cellulose is more important for the efficient natural and biotechnological conversion of cellulosic biomass through enzymes which contain these CBMs. This phenomenon is assumed to originate from the effects of changes in configurational entropy upon binding. The loss of configurational entropy is thought to be less profound upon binding to conformationally restrained insoluble cellulose, resulting in larger free energies of binding.

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ASSOCIATED CONTENT

S Supporting Information *

Ligand (CNW) preparation and characterization are described in detail. Some supplementary results are also included. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by United States Department of Agriculture-NRI: 2007-35504-18339. We are thankful for Vivek Verma from our lab for CNW preparation. Some of the CBM protein expression, purification experiments were performed in the laboratory of Dr. Ming Tien (Penn State University).

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REFERENCES

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