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Atomic Force Microscopic Study of Chitinase Binding onto Chitin and Cellulose Surfaces Yoshihiro Kikkawa,*,† Masato Fukuda,‡ Tomoya Kimura,‡ Ayumi Kashiwada,‡ Kiyomi Matsuda,‡ Masatoshi Kanesato,† Masahisa Wada,§,∥ Tadayuki Imanaka,⊥ and Takeshi Tanaka*,† †
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan ‡ Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, 1-2-1 Izumi-cho, Narashino, Chiba 275-8575, Japan § Department of Biomaterials Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan ∥ Department of Plant & Environmental New Resources, College of Life Sciences, Kyung Hee University, 1, Seocheon-dong, Giheung-ku, Yongin-si, Gyeonggi-do 446-701, Republic of Korea ⊥ Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan S Supporting Information *
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density of the enzymes on the polysaccharide surfaces.8,9 In the present study, we focus on the chitinase secreted from the hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which is composed of two catalytic domains (CatDA, CatDB) and three substrate binding domains (ChBD1, ChBD2, ChBD3).16,17 Concerning the binding domains, ChBD2 and ChBD3 have almost identical amino acid sequences (85% identity, 92% similarity within 100 amino acids), whereas ChBD1 has quite different sequence from the other ChBDs.16 Then, binding characteristics of ChBD1 and ChBD2 toward chitin and cellulose had been studied by the force curve measurements using AFM (Figure 1A); however, the analysis was conducted at a single loading rate.18,19 Therefore, the loading rate dependencies would provide the full description of the binding mechanisms of the chitinase onto polysaccharide surfaces. In this contribution, we investigate the molecular level recognition of the ChBD1/ChBD2 onto chitin/cellulose surfaces by using the AFM force spectroscopy. The equilibrium parameters of the dissociation rate constants and the width of the energy barrier are extracted from the loading rate dependence of the unbinding forces. The differences in the binding ability for ChBD1 and ChBD2 are discussed in terms of the aromatic residues in both ChBDs.
INTRODUCTION Materials production from renewable biomass has attracted much attention from the viewpoint of sustainability and low environmental impact.1,2 The biomass-derived materials can be used not only as the polymeric materials directly purified from the natural resources but also as the products biologically or chemically modified from the biomass-derived compounds.3−6 Among many kinds of biomass-derived polymers, polysaccharides are well-known as one of the most abundant natural polymers, and they can be degraded by the corresponding polysaccharide-degrading enzymes whose reactions are highly regulated by the molecular recognition process, namely, the substrate specificity of the enzymes.7−9 Specific molecular recognitions between biomacromolecules are fundamental for a living system, and among them, the carbohydrate-aromatic interactions often play one of the key roles in the biological process.10,11 Structural analysis based on the NMR and X-ray revealed the existence of CH/π interaction of CH groups in the sugar and aromatic amino acids in the carbohydrate binding sites of the enzyme.12,13 High level ab initio calculations suggest that the binding energy of CH/π interaction in the carbohydrate-aromatic system is almost the same level of the normal hydrogen bonding.14 However, the CH/π interaction has less directionality because the dispersion interaction is the major origin of the attractive interaction between the nonpolar surface in the pyranose ring and aromatic residues, whereas the electrostatic contribution is relatively small. In addition, the carbohydrate−indole interaction is more stable than the carbohydrate-benzene system.15 Chitin, the second most abundant natural polymer following to cellulose, is one of the most fascinating sustainable polymers for the various applications, which have been developed through the appropriate enzymatic saccharification process.2,3 Understanding the enzymatic degradation by the chitinase would provide the effective saccharification process, including the substrate binding and subsequent hydrolysis reaction. Most of the polysaccharide degrading enzymes contain the substratebinding modules, which support the increase of the effective © 2014 American Chemical Society
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EXPERIMENTAL SECTION
Materials. All the chemicals unless specified were purchased from either Kanto Chemical Co., Inc., or Tokyo Chemical Industry Co., Ltd., Japan. The AFM cantilevers with different spring constants were commercially available from Olympus Co. Ltd., Japan (OMCLTR400PB-1 and OMCL-TR800PB-1). The spring constants of individual cantilevers were determined by using the force calibration cantilever (CLFC probes, Bruker, U.S.A.). Received: January 10, 2014 Revised: February 12, 2014 Published: February 14, 2014 1074
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Figure 1. Schematic drawings of interaction between the ChBDs and polysaccharide surfaces (A), chemically modified AFM tips (B), and chemical structures of chitin (C) and cellulose (D). In (A), conserved surface-exposed and aligned Tyr and Trp residues were, respectively, highlighted as blue and red color in the modeled tertiary structures of ChBDs from T. kodakarensis,18 and they interact with pyranose ring of polysaccharide via CH/π interaction. The distances between these linearly aligned aromatic residues (ca. 1 nm) were identical to the lattice constants along the crystallographic caxis in the polysaccharides of chitin and cellulose. The modification steps of AFM tips in (B), see Experimental Section.
Figure 2. Typical force curve data (A) and histograms of rupture forces between ChBD2 and chitin (B), and those between ChBD2 and cellulose (C) at the loading rate of 8 nN/s. The blue and red curves in (B) and (C) are corresponding to the FFT-smoothed and following Gaussian fitted data, respectively. The force values for intervals between Gaussian curve peaks are indicated. Immobilization of ChBDs on AFM Tips. The purification of the His-tagged ChBD1 and ChBD2 from Escherichia coli cells and immobilization on the AFM tip were performed according to the previous method.18 Briefly, the AFM cantilever was thoroughly cleaned by using UV-ozone cleaner (UV253, Filgen) and immersed in the 1 mM ethanol solution of HS(CH 2 ) 1 1 (OCH 2 CH 2 ) 6 OCH 2 COOH (PEG6COOH; Prochimia, U.S.A.). The terminal carboxylic acid groups were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in order to connect with N-(5-amino-1-carboxypentyl) iminodiacetic acid (NTA; Dojindo, Japan) through an amide bond. The residual NHS ester groups were inactivated with 1 M ethanolamine solution. The metalation of the AFM tip modified with NTA groups was performed in 0.1 M Ni(II)SO4
dissolved in 0.1 M Tris·HCl (pH 7.5). Finally, the AFM tip was functionalized in 0.1 M Tris·HCl (pH 7.5) containing 3 μg/mL Histagged ChBD1 or ChBD2 (Figure 1B). Preparation of Chitin and Cellulose Substrates. The chitin thin film was prepared by spin-cast method on a silicon support cleaned with UV-ozone cleaner. β-Chitin (Seikagaku Corporation, Japan) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, and the solution was spin-cast on the silicon supports at a rotation speed of 3000 rpm. Cellulose films from a green alga Cladophora sp. prepared by a reported method20 were fixed on a silicon support (1 × 1 cm) using a waterresistant glue. These chitin and cellulose substrates were fixed on an AFM liquid cell. 1075
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AFM Force Spectroscopy. The force curve between the chitin/ cellulose substrates and His-tagged ChBDs was measured by using an AFM (SPI4000/SPA400; SII nanotechnology Inc., Japan). All the AFM experiments were performed in a 0.1 M Tris·HCl buffer solution (pH 7.5). The force curves were recorded with different loading rates ranging from about 1 to 100 nN/s. Over 1000 data were collected by using different tips and chitin/cellulose substrates for the histogram analysis. All the force data were analyzed by SPIP software (Image Metrology, Denmark).
As shown in Figure 3, the most probable pull-off forces increased linearly with an increase in the logarithm of loading
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RESULTS AND DISCUSSION AFM force spectroscopy was performed by using the ChBD2 functionalized AFM tips onto either chitin or cellulose surface in a Tris·HCl buffer solution. Figure 2A shows the typical force curves with and without ChBD2 on the AFM tip. No peaks were found in the control experiment using the modified AFM tips terminated with NTA groups. In contrast, ChBD2 functionalized AFM tip revealed the multiple peaks corresponding to the rupture force between the ChBD2 and chitin/cellulose surfaces. The frequency of the force curves showing multiple pull-off events was about 20−30% of total force measurements (n ≥ 1000) at the loading rate of 8 nN/s. This result suggests that there is less nonspecific interaction of the ligands used as a linker and that ChBD2 can bind onto both chitin and cellulose surfaces. The single binding force of ChBD2 was extracted from the histograms composed of a number of force curve data (n ≥ 200), as shown in Figure 2B,C. Fast Fourier Transform (FFT) smoothening and following Gaussian function fitting allowed to find out the local maxima in the data. The intervals between red peaks in Figure 2B,C can be corresponding to a rupture force of a single enzyme onto polysaccharide surfaces. For example, at the loading rate of 8 nN/s, the binding force of the ChBD2 onto chitin was measured to be 107 ± 20 pN, whereas that onto cellulose was 110 ± 24 pN. This result indicates that the rupture force of ChBD2 onto chitin is almost identical to that onto cellulose. This was the case at the loading rate of 2 nN/s, as reported in the previous studies.18,19 The most commonly used theoretical model for the single force spectroscopy, namely, Bell-Evans model21,22 has predicted a linear relationship between the most probable binding force (F) and the logarithm of the loading rate (r), allowing the calculation of the effective bond length (xβ) and dissociation rate constants (kd) from the following equation: ⎛ ⎞ r ⎟ ⎜ F = fβ ln⎜ ⎟ ⎝ f β kd ⎠
Figure 3. Loading rate dependence of the pull-off forces between ChBDs and chitin/cellulose surfaces. The force data were obtained from the histogram analysis on the basis of the Gaussian fit to the histograms with different loading rates. The dashed-lines for ChBD2 are a numerical fit of experimental data to the Bell-Evans model.
rate. The linear regression yielded xβ = 0.14 ± 0.02 nm and kd = (4.4 ± 0.3) × 10−3 s−1 for chitin, and xβ = 0.14 ± 0.02 nm and kd = (3.7 ± 0.2) × 10−3 s−1 for cellulose on the basis of eqs 1 and 2. These values for chitin were almost identical to those for cellulose, suggesting that ChBD2 can bind onto both chitin and cellulose in a similar manner even if the side chain groups of the polysaccharide are different. The main driving force for the ChBD2 binding can be carbohydrate−aromatic interactions composed of the CH/π interaction between the CH groups of the pyranose ring in polysaccharide and π electron density of surface exposed aromatic residue in ChBD2 (Trp-636, Trp-669, and Trp-687, Figure 1A). In the present force analyses, it could be confirmed that both xβ and kd of ChBD2 onto chitin/cellulose surfaces are almost identical, and these values are not influenced by the side chain groups of the polysaccharides. In our previous report,18 a simplified binding model was proposed on the basis of the comparison of binding forces between ChBD1 and ChBD2, which were proportional to the number of the conserved, surface-exposed, and aligned aromatic residues of the ChBDs at the relatively low loading rate of 2 nN/s (see Figure 1A). The question has arisen whether this relationship is applicable for the whole range of loading rates or not. Therefore, the force measurements for ChBD1 were also examined for the higher loading rates (see SI), and plotted in Figure 3. Unexpectedly, the binding forces for ChBD1 onto chitin/cellulose were ca. 2/3 of those for ChBD2 within the range of 1−10 nN/s, but they were out of the 2/3 rules and much smaller than the force values for ChBD2 in the range of 10−100 nN/s. The interaction sites of ChBD1 are Tyr-110 and Trp-111, whereas those of ChBD2 are Trp-636, Trp-669, and Trp-687,18 as shown in Figure 1A. It has been reported that binding affinity between the hevein (an allergen protein) and chitooligosaccharide are modulated depending on the amino acid residues of Tyr and Trp in the enzyme.28 The original hevein preserves the two Trp residues in the binding site and shows the highest affinity, whereas mutation from Trp to Tyr strongly suppressed the intermolecular interaction. Taking the interaction sites of ChBD1 and ChBD2 into account, it can be proposed that the binding interaction of ChBD1 containing Tyr and Trp cannot
(1)
Then, fβ is given by
fβ =
kBT xβ
(2)
where kB is the Boltzmann constant and T is the temperature. Thus, many biological receptor−ligand systems have been analyzed for the evaluation of the interactions of ligand−receptor pairs.21−27 Therefore, we also applied the Bell-Evans model to further compare the binding abilities of ChBD2 onto chitin/ cellulose surfaces. Force curve measurements were repeated at different loading rates to determine the pull-off force dependence on the loading rate (see Supporting Information). Then, the rupture forces of ChBD2 were plotted as a function of loading rate to convert the force data to equilibrium parameters on the basis of the Bell-Evans model. 1076
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ACKNOWLEDGMENTS The authors thank Dr. Masahiro Fujita (RIKEN, Japan) for the discussion of force data. This work has been partly supported by the grant from JSPS (23750137) and MEXT KAKENHI (23106722), Japan.
hold enough relaxation time at the higher loading rate than that of ChBD2 possessing three Trp residues. In other words, all the Trp residues in the ChBD2 simultaneously work just like a single binding site at the whole range of loading rates. In contrast, both Tyr and Trp residues in ChBD1 bind onto the substrates at the lower loading rates, whereas only Trp residue participate in the binding event especially at the higher loading rate because Tyr residue without sufficient interaction time can be pulled apart before the firm substrate binding. Therefore, the force values of ChBD1 are apparently almost constant over the present experimental condition, namely, the Tyr residue is less active and more labile at the high loading rate than the Trp one. Thus, AFM force spectroscopy analyses revealed the different binding manners of ChBD1 and ChBD2 onto chitin/cellulose surfaces and provided the insight into the recognition of polysaccharide surfaces by the ChBDs in the molecular level. Polysaccharide-binding modules work in the initial step of the enzymatic degradation reaction, and in most cases, the bound enzymes collapse the crystalline regions of the polysaccharides for the effective enzymatic hydrolysis reaction.29−31 Since the present study revealed the similar ChBDs’ binding ability onto both chitin and cellulose, the disruption of the crystalline regions of cellulose may also be possible even by the ChBDs. Therefore, it can be proposed that combinations of thermally stable ChBDs and endotype cellulase have potential application for the effective saccharification of cellulose to produce useful biomass-derived chemicals.
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CONCLUSION A number of force curve analyses were performed to study the effect of loading rate on the unbinding force between ChBDs and chitin/cellulose surfaces. The pull-off force of ChBD2 was linearly increased with an increase in the logarithm of loading rate, whereas that of ChBD1 was almost constant at the range of 1 to 100 nN/s. The xβ and kd of ChBD2 onto chitin/cellulose surfaces were almost identical, suggesting the CH/π interaction between the aromatic residues in the ChBD2 and pyranose ring of polysaccharide is the major interaction and that the side chain groups of the polysaccharide do not affect the binding event. In contrast, the force data of ChBD1 could not be analyzed on the basis of the Bell-Evans model. The amino acid residues participating in the binding event for ChBD1 are Tyr and Trp, whereas those for ChBD2 are three Tyr residues, resulting in the different binding manners between the ChBD1 and ChBD2 onto polysaccharide surfaces. ASSOCIATED CONTENT
S Supporting Information *
The histogram analyses to calculate the rupture forces between ChBDs and chitin/cellulose surfaces at various loading rates. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +81-29-861-2955. Fax: +81-29-861-3029. E-mail: y.
[email protected]. *Phone: +81-29-861-2903. Fax: +81-29-861-2786. E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 1077
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