Interaction Force of Chitin-Binding Domains onto Chitin Surface

Interaction force of chitin-binding domains (ChBD1 and ChBD2) from a ... (10) Among the three ChBDs, ChBD1 is located near the N-terminus of the chiti...
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Interaction Force of Chitin-Binding Domains onto Chitin Surface Yoshihiro Kikkawa,*,† Hideo Tokuhisa,† Hajime Shingai,‡ Tomohiro Hiraishi,§ Hirohiko Houjou,‡ Masatoshi Kanesato,† Tadayuki Imanaka,| and Takeshi Tanaka*,# Nanoarchitectonics Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8562, Japan, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan, Bioengineering Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan, and Nanotechnology Research Institute, AIST, Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8562, Japan Received February 13, 2008; Revised Manuscript Received May 15, 2008

Interaction force of chitin-binding domains (ChBD1 and ChBD2) from a thermostable chitinase onto chitin surface was directly measured by atomic force microscopy (AFM) in a buffer solution. In the force curve measurement, multiple pull-off events were observed for the AFM tips functionalized with either ChBD1 or ChBD2, whereas the AFM tips terminated with nitrilotriacetic acid groups without ChBD showed no interaction peak, suggesting that the detected forces are derived from the binding functions of ChBDs onto the chitin surface. The force curve analyses indicate that the binding force of ChBD2 is stronger than that of ChBD1. This result suggests that ChBD1 and ChBD2 play different roles in adsorption onto chitin surface.

Introduction Chitin is a member of polysaccharide composed of Nacetylglucosamine units, which are covalently connected by β-1,4 linkages. Chitin can be found in the natural environment, and is the main component of the cell walls of fungi and the exoskeletons of arthropods. Chitin is degraded by chitinases secreted from microorganisms such as bacteria, fungi and archaea.1–9 In this study, we focused on the chitinase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.6 The chitinase is organized with five characteristic domains, namely, dual catalytic domains (CatDs) and triple chitin-binding domains (ChBDs), as shown in Figure 1. The two CatDs show the different cleavage specificities, that is, CatDA and CatDB carry out the exo- and endotype hydrolysis reaction, respectively.10 Among the three ChBDs, ChBD1 is located near the N-terminus of the chitinase, and ChBD2 and ChBD3 existing near the center position have almost identical amino acid sequences (85% identity within 100 amino acids). It has been reported that ChBD2, ChBD3, and two CatDs are connected by flexible linkers (43-56 amino acids residues).6 All the ChBDs are probably functional for adsorption onto chitin surface to accumulate the CatDs. ChBD1 and ChBD2 (or ChBD3) should have differential binding interaction because their amino acid sequences are quite different. However, the functions of individual ChBDs have not experimentally demonstrated yet, and binding force as well as adsorption roles for each ChBD has remained unclear. * Corresponding authors. Phone: +81-29-861-2955(Y.K.); +81-29-8612903(T.T.). Fax: +81-29-861-3029 (Y.K.); +81-29-861-2786 (T.T.). E-mail: [email protected] (Y.K.); [email protected] (T.T.). † Nanoarchitectonics Research Center, AIST. ‡ University of Tokyo. § RIKEN Institute. | Kyoto University. # Nanotechnology Research Institute, AIST.

Atomic force microscopy (AFM) has been recognized as one of the powerful tools for not only the analysis of surface morphologies and properties but also the measurement of individual molecular interactions as a force spectroscopy. By modifying the AFM tip with specific chemicals or enzymes, interaction forces between the complimentary receptor-ligand systems could be directly resolved, such as DNA strands,11 antibody-antigens,12–14 cell adhesion molecules,15,16 proteinDNA,17,18 supramolecular host-guest complexes,19–22 polyesterenzyme,23 and so on. Therefore, force curve measurement using AFM tips functionalized with individual ChBDs would enable us to directly probe the binding force of ChBDs onto the chitin surface. In this contribution, we demonstrate that individual ChBD1 and ChBD2 can adsorb onto the chitin surface and study their binding interaction by AFM for the first time. The AFM tip was modified by the ligand containing hexa(ethylene glycol) and nitrilotriacetic acid (NTA) groups. Subsequently, the tip was functionalized with His-tagged ChBD1 or ChBD2 through the coordination bonds of NTA and Ni(II). Direct measurement of binding force was performed by AFM in a buffer solution. The mechanism of chitin degradation by chitinase from T. kodakaraensis KOD1 was developed on the basis of the difference between the binding force of ChBD1 and that of ChBD2.

Experimental Section Construction of Expression Plasmids. Escherichia coli JM109 and BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA) were used as hosts for the expression plasmid and were cultivated in LB medium at 37 °C. DNA manipulations were carried out by standard methods, as described by Sambrook and Russell.24 Restriction enzymes and other modifying enzymes were purchased from Takara Bio (Otsu, Shiga, Japan) or Toyobo (Osaka, Japan).

10.1021/bm800162x CCC: $40.75  2008 American Chemical Society Published on Web 07/26/2008

Chitin-Binding Domains onto Chitin Surface

Figure 1. (a) Domain structure of the chitinase from T. kodakaraensis KOD1. Amino acid sequence of His-tagged ChBD1 and ChBD2, (b) and (c), respectively. Underlined region denotes the His-tag, whereas shaded regions exhibit the ChBD sequences. The numbers beside sequences indicate the number of the amino acid residues in original chitinase. The sequences of SSGLVPRGSHM are linker peptides derived from expression vector. (d) Chitin-binding experiment assessed by SDS-PAGE. Lanes 1-3 for ChBD1 and lanes 4-6 for ChBD2. Lanes 1 and 4, the purified ChBDs before binding experiment; lanes 2 and 5, unbound fractions after binding experiment; lanes 3 and 6, bound fractions onto colloidal chitin; and lane M, the molecular mass standards.

The expression plasmid for His-tagged ChBD1 was constructed by PCR as described below. Two oligonucleotides (F1, 5′-CTGTTCCATATGAACAACCTGCTGGTGGTCG-3′; R1, 5′-TTAGATCTCAGACAACATCCCCTACGAGCTC-3′ [underlined sequences indicate NdeI and BglII sites in the F1 and R1 primers, respectively]) and T. kodakaraensis genomic DNA were used as primers and template for DNA amplification, respectively. The amplified DNA was digested with NdeI and BglII and then ligated with the NdeI and BamHI sites in the plasmid pET-15b (Novagen, Madison, WI). The resulting plasmid was designated as pET-ChBD1. For His-tagged ChBD2 construction, a set of oligonucleotides (F2, 5′-CTCCACATATGGGGGATTTTGTCAAGCCGGGTTCT-3′;R2,5′-GTGGATCCTCACCAGGCATCCCAGAGCTGGC-3′ [underlined sequences indicate NdeI and BamHI sites in the F2 and R2 primers, respectively])) were used for PCR, and the resulting plasmid was designated as pET-ChBD2. Purification of Recombinant ChBDs. E. coli BL21-CodonPlus(DE3)RIL cells harboring pET-ChBD1 or pET-ChBD2 were induced for overexpression with 0.1 mM isopropyl-β-D-thio-galactopyranoside at the midexponential growth phase and incubated for a further 3.5 h at 37 °C. The cells were harvested by centrifugation (5000 × g for 10 min at 4 °C), resuspended in buffer A (50 mM Tris-HCl (pH ) 7.5)), and then disrupted by sonication. The supernatant after centrifugation (12000 × g for 30 min) was incubated at 80 °C for 20 min and centrifuged (12000 × g for 15 min) to obtain a heat-stable protein solution. Ammonium sulfate precipitation of the solution was performed at 40% saturation for ChBD1 or 60% for ChBD2. The precipitate was dissolved in buffer A, and the solution was applied to an affinity

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HisTrap column (1 mL; GE healthcare, U.K.) equilibrated with buffer A containing 0.3 M NaCl. The proteins were eluted with buffer A containing 0.3 M NaCl and 0.5 M imidazole, and the fractions containing ChBDs were desalted using a Hitrap-desalting column (5 mL; GE healthcare). Protein concentration was determined with the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. Chitin-Binding Experiment. Binding experiment of ChBDs and preparation of colloidal chitin were performed according to the previous method.6 The purified ChBDs were mixed with the colloidal chitin as a substrate, and the mixture was incubated for 1 h at room temperature (25 °C). Then, the solution was centrifuged to separate the supernatant containing unbound ChBDs from the colloidal chitin, onto which there are bound ChBDs. The precipitates were washed with a buffer solution. The supernatant and precipitates were assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Preparation of Chitin Thin Films. All the chemicals unless specified were purchased from Kanto Chemical Co., Inc. or Tokyo Chemical Industry Co., Ltd., Japan. Thin film of chitin was prepared by spin-cast method on a silicone wafer cleaned with ultraviolet-ozone cleaner (Nippon Laser & Electronics Laboratory, Japan). β-Chitin (Seikagaku Corporation, Japan, the nominal molecular weight is ca. 2 × 105) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol with vigorous stirring. The 0.5% (w/v) solution was spin-cast on the silicon supports at a rotation speed of 3000 rpm, resulting in the formation of the thin films of about 50 nm thickness. Functionalization of AFM Tip. A gold-coated AFM cantilever (Olympus Co. Ltd., Japan, OMCL-TR400PB-1) was used for the force curve measurements. The spring constant of the cantilever was calibrated according to the method by Hazel and Tsukruk25 and confirmed to be about 0.09 N/m, which was in good agreement with the reported value.20 The procedure of functionalized AFM cantilever tip is summarized in Supporting Information. All the reactions were checked on the gold-coated glass substrates by using Fourier transformed infrared external reflection spectroscopy (FTIR-ERS; Nicolet Magna 760, U.S.), as shown in Supporting Information. AFM Analysis. Morphological observation and force spectroscopy analysis were carried out by using AFM (SPI4000/SPA400; SII Nanotechnology Inc. Japan,) in 0.1 M Tris-HCl buffer solution (pH ) 7.5). The silicon cantilever (Olympus Co. Ltd., Japan, OMCL-AC240TSC2, spring constant ) 2 N/m) was operated in a dynamic force (tapping) mode to observe the surface morphology of the chitin thin film. For the force curve measurement, the cantilever modified by the above method was used. The force curve was obtained with the loading rate of 20 nm/s,26 and analyzed by the SPIP software (Image Metrology, Denmark). The force-displacement curve was converted to force-distance curve. More than 500 force curve measurements were performed for AFM tips terminated with NTA groups as well as ChBDs. The force curves with multiple pull-off events were only used for the analysis, and the last pull-off force was used to make histograms. Smoothening by the Fast Fourier Transform (FFT) filtering and subsequent fitting of Gaussian peaks were conducted to search the center position of local maxima.

Results To analyze the different types of ChBDs in the chitinase from the hyperthermophilic archaeon T. kodakaraensis, we first prepared His-tagged ChBD1 and ChBD2. Recombinant ChBDs were produced by conventional genetic engineering methods using E. coli system and purified (Figure 1d, lanes 1 and 4). The observed molecular mass was consistent with the deduced mass of each ChBD. We then performed chitin-binding experiment to confirm the adsorptive ability of the ChBDs (Figure 1d). Although both ChBDs were detected also at unbound fractions (lanes 2 and 5), it was clarified that they adsorbed onto chitin because they remained at bound fractions even after

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Figure 2. (a) AFM height image of β-chitin spin-coated on Si wafer. The color contrast of the image is a total range of 20 nm. (b–d) Retraction curves obtained by the force curve measurement using different AFM tips in the buffer solution. (b) NTA terminated; (c) functionalized by Histag ChBD1; (d) His-tag ChBD2 AFM tip. (e and f) Histograms of pull-off forces for ChBD1 and ChBD2, respectively.29 The histogram data was FFT-smoothed (blue line), and Gaussian curves were fitted to the histograms (red lines). The force values for each of the Gaussian curve peaks are indicated.

washing with the buffer (lanes 3 and 6). Comparing between the relative stained band density of bound and unbound ChBDs in the gel, greater amount of bound fragments were found in ChBD2 than unbound ones. However, in the case of ChBD1, the result was opposite, that is, the bound fragments were smaller amount than the unbound ones. This result suggests that both ChBDs have the ability to bind onto the colloidal chitin surface, and that ChBD2 has the stronger activities in binding onto colloidal chitin than ChBD1 within the present experimental condition. For the study of binding activities of ChBDs onto chitin surface directly, the force curve measurements by AFM would be appropriate because it allows us to determine the binding force. Prior to the force curve measurements, chitin thin film was prepared by spin-cast method, and the surface morphology was observed by AFM in the buffer. Figure 2a shows the AFM image of the β-chitin thin film cast on a silicon wafer (see Figure S3 in Supporting Information). β-Chitin has a highly crystalline fibriller structure,27 and AFM observation revealed that the surface of thin film is covered with randomly stacked chitin crystals.

The force-distance curve was measured in the buffer. Only the representative retraction curves are shown in Figure 2b-d. First, a negative control experiment was peformed by using the modified AFM cantilever tip terminated with NTA groups, which has no ChBDs, and the result is shown in Figure 2b. No significant signal was observed. Such force curves could be received with the frequency above 96%, suggesting that specific interaction does not exist in this system possibly due to the existence of hexa(ethylene glycol) unit, which is often used to prevent the nonspecific interaction,28 on the modified AFM tip. In contrast, AFM tip functionalized with His-tagged ChBD1 or ChBD2 showed the multiple rupture events, as shown in Figure 2c,d. Such events were observed in the force curve with the frequency of 50% for ChBD1 and 39% for ChBD2. Because the AFM tip modified with only NTA showed no pull-off force event, the multiple rupture events observed in Figure 2c,d are derived from the specific interaction between the chitin and ChBDs. To analyze the binding force for a single ChBD molecule, histogram analysis was perfomed. To this end, 270 force curve data with multiple rupture events were collected for ChBD1

Chitin-Binding Domains onto Chitin Surface

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Figure 3. Proposed mechanism of chitin degradation by the chitinase from T. kodakaraensis KOD1 on the basis of the binding interaction force of ChBDs and hydrolysis manners of CatDs.10

and ChBD2, respectively. The data are summarized as histograms in Figure 2e,f. Against the histograms, FFT smoothening and following Gaussian function fitting was performed to find the local maxia in the data, which is related to the rupture of a single receptor-ligand system.19 Almost equivalent intervals of the local maximum forces were detected in each histogram. Subtraction of the neighboring local maximum forces provided the binding force of single ChBD. In the case of ChBD1, the binding force of a single moleucle was about 60 pN, whereas that for ChBD2 was about 90 pN.29 This result indicates that binding force of ChBD2 is 1.5 times stronger than that of ChBD1.

Discussion In this study, we prepared recombinant ChBD1 and ChBD2 without CatDs and confirmed the binding ability of ChBD itself to chitin (Figure 1). Then, the ChBDs were attached onto the AFM tip, and the binding forces between ChBDs and chitin surface were

directly measured by force spectroscopy. From hundreds of measurements and their statistical analyses, we clarified the binding force of single ChBD molecule (Figure 2). It is known that ChBDs bind to chitin via surface-exposed aromatic amino acid redsidues.30 The aromatic rings lie lineary in the same plane of protein surface and directly stack pyranose rings of sugar chains by hydrophobic interaction. We performed homology modeling of ChBD1 and ChBD2 and found that two and three conserved aromatic residues aligned on the surfaces of ChBD1 and ChBD2, respectively (see Figure S5 in Supporting Information). From the viewpoint of the numbers of the aromatic residues, it can be expected that the binding force of ChBD2, which retains the three aromatic residues, is greater than that of ChBD1, which does the two. This estimation agrees well with the result of the binding force analysis obtained here that the binding force of single ChBD2 (ca. 90 pN) is stronger than that of ChBD1 (ca. 60 pN).

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It has been reported that CatDA near the ChBD1 catalyzes the hydrolysis of chitin as exotype cleavage, whereas the CatDB near the ChBD2 and ChBD3 acts as an endotype (Figure 1), and proposed that concerted actions of these CatDs enable the effective chitin degradation from the viewpoint of cleavage specificities.10 In this study, we addressed the individual functions of ChBDs and found that the binding force of ChBD2 is stronger than that of ChBD1. This result suggests that ChBD1 and ChBD2 play different roles in adsorption onto chitin surface. Taking the cleavage specificities of CatDs and the binding force of ChBDs into account, we would like to further develop the mechanism of chitin degradation by the multidomain chitinase from T. kodakaraensis KOD1 (Figure 3.) At an initial step, binding event onto the chitin surface takes place by the function of ChBD2 and ChBD3. Synergetic effect of binding affinity by dual binding domains has been found in not only chitinase31 but also cellulase,32 xylanase,33 and polyhydroxyalkanoate (PHA) depolymerase.34 The ChBD3 has almost the same amino acid sequence as ChBD2, suggesting that the binding interaction force of the ChBDs is totally above twice as much as single ChBD2 (binding force > 180 pN). Such synergetic binding interaction of ChBD2 and ChBD3 results in the strong adsorption against the chitin surface to secure a footing for the following degradation steps. At this stage, disturbance of the chitin crystal structure,35 which facilitates the subsequent hydrolysis reaction by CatD, should occur due to such an strong binding interaction, as similar to the binding domain of cellulase36 and PHA depolymerase.37,38 Then, the endotype action of CatDB produces the molecular chain ends of chitin through the hydrolysis. The chain ends created in the former degradation step were hydrolyzed by exotype CatDA, which produces chitin dimer.10 During the exotype degradation, N-terminal ChBD1 directly linked to CatDA probably plays an important role, namely, the ChBD1 binds and guides the chitin chains into active site of CatDA, as reported in Serratia chitinase B, which retain ChBD homologus to ChBD1.39 Due to the relatively weak binding force (ca. 60 pN), the ChBD1 can continue the attachment and detachment near the chain ends and effectively introduces the increasing numbers of chain ends to CatDA for the consecutive exotype hydrolysis. Thus, the chitinase continues the hydrolysis reaction with the cooperative action of ChBDs and CatDs.

Conclusions Recombinant ChBD1 and ChBD2 of the chitinase from the hyperthermophilic archaeon T. kodakaraensis were prepared to study the binding interaction onto chitin. By the chitin-binding experiment, it was confirmed that both ChBDs have the ability to bind onto chitin. Force curve measurement by AFM directly revealed the interaction force of individual ChBD1 and ChBD2 onto chitin surface. The binding force of single ChBD1 was estimated as ca. 60 pN, whereas that of ChBD2 was ca. 90 pN, namely, ChBD2 has 1.5 times stronger binding force than ChBD1. This result suggests the different roles between ChBD1 and ChBD2 in binding onto chitin surface. The mechanism of chitin degradation by the chitinase from T. kodakaraensis KOD1 was developed on the basis of the difference in the binding force between ChBD1 and ChBD2. Acknowledgment. This work was partly supported by the grant from the Foundation Advanced Technology Institute (T.T.). We would like to thank Ms. Satoko Ishibe for technical assistance in FT-IR measurement.

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Supporting Information Available. The procedure of functionalized AFM cantilever tip, determination of AFM tip modifications by FTIR-ERS, structural characterization of chitin film, histogram analysis of ChBD2, and homology modeling of ChBD1 and ChBD2. This material is available free of charge via the Internet at http://pubs.acs.org.

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