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Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose*

  • Akihiko Nakamura
    Affiliations
    From the Okazaki Institute for Integrative Bioscience and

    the Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (SOKENDAI), Kanagawa 240-0193, Japan,
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  • Tomoyuki Tasaki
    Affiliations
    the Department of Applied Chemistry, Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan,
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  • Daiki Ishiwata
    Affiliations
    From the Okazaki Institute for Integrative Bioscience and
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  • Mayuko Yamamoto
    Affiliations
    From the Okazaki Institute for Integrative Bioscience and
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  • Yasuko Okuni
    Affiliations
    From the Okazaki Institute for Integrative Bioscience and
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  • Akasit Visootsat
    Affiliations
    the Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand,
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  • Morice Maximilien
    Affiliations
    the National Chemical Engineering Institute in Paris, Paris 75005, France,
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  • Hiroyuki Noji
    Affiliations
    the Department of Applied Chemistry, Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan,
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  • Taku Uchiyama
    Affiliations
    the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan, and
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  • Masahiro Samejima
    Affiliations
    the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan, and
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  • Kiyohiko Igarashi
    Affiliations
    the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan, and

    the VTT Technical Research Centre of Finland, Espoo FI-02044 VTT, Finland
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  • Ryota Iino
    Correspondence
    To whom correspondence should be addressed: Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi 444-8787, Japan. Tel.: 81-564-59-5230; E-mail: .
    Affiliations
    From the Okazaki Institute for Integrative Bioscience and

    the Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (SOKENDAI), Kanagawa 240-0193, Japan,

    Institute for Molecular Science, National Institutes of Natural Sciences, Aichi 444-8787, Japan,
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  • Author Footnotes
    * This work is supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology 16H00789, 16H00858, and 15H04366 (to R. I.) and 15H06898 (to A. N.); Imaging Science Project of the Center for Novel Science Initiatives, National Institutes of Natural Sciences Grants IS271006 and IS281005 (to R. I.), and ORION project of Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences (to R. I.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:September 08, 2016DOI:https://doi.org/10.1074/jbc.M116.752048

      Abstract

      Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Iα. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30–40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.

      Introduction

      Cellulose, a major component of plant cell walls, is the most abundant biopolymer on earth and has potential as a regeneratable source of bioenergy and chemicals (
      • Himmel M.E.
      • Ding S.Y.
      • Johnson D.K.
      • Adney W.S.
      • Nimlos M.R.
      • Brady J.W.
      • Foust T.D.
      Biomass recalcitrance: engineering plants and enzymes for biofuels production.
      ,
      • Chundawat S.P.
      • Beckham G.T.
      • Himmel M.E.
      • Dale B.E.
      Deconstruction of lignocellulosic biomass to fuels and chemicals.
      ). Cellulases from microorganisms are used to degrade physically and chemically stable crystalline celluloses into oligosaccharides under mild conditions (
      • Payne C.M.
      • Knott B.C.
      • Mayes H.B.
      • Hansson H.
      • Himmel M.E.
      • Sandgren M.
      • Ståhlberg J.
      • Beckham G.T.
      Fungal cellulases.
      ,
      • Wilson D.B.
      Microbial diversity of cellulose hydrolysis.
      ). Among cellulases, cellobiohydrolase (CBH)
      The abbreviations used are: CBH, cellobiohydrolase; TrCel7A, T. reesei Cel7A; TrCel6A, T. reesei Cel6A; CBM, cellulose binding module; CD, catalytic domain; GH, glycoside hydrolase; HS-AFM, high speed atomic force microscopy; PASC, phosphoric acid-swollen cellulose.
      directly hydrolyzes crystalline cellulose into cellobiose. The fungal CBHs Trichoderma reesei Cel7A (TrCel7A) and Cel6A (TrCel6A) are the most studied CBHs.
      The previous studies proposed the processive hydrolysis of TrCel7A and TrCel6A, a continuous reaction without dissociation from cellulose surface. Electron microscopic observations revealed that the edges of crystalline cellulose were sharpened after treatment with TrCel7A and TrCel6A, suggesting the processive hydrolysis from the chain ends (
      • Imai T.
      • Boisset C.
      • Samejima M.
      • Igarashi K.
      • Sugiyama J.
      Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals.
      ,
      • Chanzy H.
      • Henrissat B.
      Undirectional degradation of valonia cellulose microcrystals subjected to cellulase action.
      ). Furthermore, it has been shown that the unique tunnel-shaped catalytic site keeps the enzyme bound on the substrate after each hydrolytic event, which enables processive hydrolysis and movement on crystalline cellulose (
      • Kipper K.
      • Väljamäe P.
      • Johansson G.
      Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as “burst” kinetics on fluorescent polymeric model substrates.
      ). The processive reaction has been thought to be a key for the efficient cellulose degradation. However, it is not easy to directly prove the processive hydrolysis and movement of TrCel7A and TrCel6A by biochemical assays and electron microscopy observations.
      Single-molecule imaging of biomolecules is a powerful method to visualize the elementary steps of reactions and mechanical motions of enzymes and motor proteins (
      • Bustamante C.
      • Chemla Y.R.
      • Forde N.R.
      • Izhaky D.
      Mechanical processes in biochemistry.
      ,
      • Joo C.
      • Balci H.
      • Ishitsuka Y.
      • Buranachai C.
      • Ha T.
      Advances in single-molecule fluorescence methods for molecular biology.
      • Greenleaf W.J.
      • Woodside M.T.
      • Block S.M.
      High-resolution, single-molecule measurements of biomolecular motion.
      ). The processive movement of TrCel7A on crystalline cellulose has been directly proved by single-molecule imaging with high speed atomic force microscopy (HS-AFM), and the translational rate constant (ktr) has been successfully estimated (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ,
      • Igarashi K.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Penttilä M.
      • Samejima M.
      High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose.
      ). Very recently, even the step size (∼1 nm) of the movement was analyzed with single-molecule measurements using optical tweezers (
      • Brady S.K.
      • Sreelatha S.
      • Feng Y.
      • Chundawat S.P.
      • Lang M.J.
      Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.
      ). However, the processive movement of TrCel6A has not been demonstrated (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ), and it is unclear whether TrCel6A can processively move on crystalline cellulose. Because external force applied by the cantilever of the HS-AFM is one possible factor preventing the movement of TrCel6A, single-molecule methods without perturbation by external force are required to verify this issue.
      In addition to the processive movement (hydrolysis), the overall cycle of the reaction of CBH includes binding and dissociation on crystalline cellulose. The binding rate constant (kon) and dissociation rate constant (koff) for intact TrCel7A against crystalline cellulose were successfully estimated using single-molecule fluorescence imaging (
      • Shibafuji Y.
      • Nakamura A.
      • Uchihashi T.
      • Sugimoto N.
      • Fukuda S.
      • Watanabe H.
      • Samejima M.
      • Ando T.
      • Noji H.
      • Koivula A.
      • Igarashi K.
      • Iino R.
      Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.
      ,
      • Jung J.
      • Sethi A.
      • Gaiotto T.
      • Han J.J.
      • Jeoh T.
      • Gnanakaran S.
      • Goodwin P.M.
      Binding and movement of individual Cel7A cellobiohydrolases on crystalline cellulose surfaces revealed by single-molecule fluorescence imaging.
      • Nakamura A.
      • Watanabe H.
      • Ishida T.
      • Uchihashi T.
      • Wada M.
      • Ando T.
      • Igarashi K.
      • Samejima M.
      Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.
      ). However, the kon and koff for TrCel6A have not been estimated at the single-molecule level. Furthermore, although both TrCel7A and TrCel6A contain the cellulose binding module (CBM), catalytic domain (CD), and glycosylated linker region connecting the CBM and CD, the role of each domain on binding and dissociation is not fully understood yet (
      • Reinikainen T.
      • Teleman O.
      • Teeri T.T.
      Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei.
      ,
      • Tomme P.
      • Van Tilbeurgh H.
      • Pettersson G.
      • Van Damme J.
      • Vandekerckhove J.
      • Knowles J.
      • Teeri T.
      • Claeyssens M.
      Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis.
      ). Previous biochemical studies showed that the CBM increases the amount of enzyme bound on the cellulose, indicating that the main role of the CBM is to increase the affinity to cellulose (
      • Stahlberg J.
      • Johansson G.
      • Pettersson G.
      A new model for enzymatic hydrolysis of cellulose based on the two-domain structure of cellobiohydrolase I.
      ). However, most previous studies only estimated the dissociation constant (Kd) in equilibrium, and the kon and koff are not reported, except for the study by Carrand and Linder in 1999 (
      • Carrard G.
      • Linder M.
      Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei.
      ). In addition, although high affinity binding of the glycosylated linker region of TrCel6A to the cellulose surface has been predicted by a computational analysis (
      • Payne C.M.
      • Resch M.G.
      • Chen L.
      • Crowley M.F.
      • Himmel M.E.
      • Taylor 2nd, L.E.
      • Sandgren M.
      • Ståhlberg J.
      • Stals I.
      • Tan Z.
      • Beckham G.T.
      Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose.
      ), experimental evidence has not been reported yet. To understand how CBHs efficiently hydrolyze crystalline cellulose on the liquid-solid interface, the rate constants of binding and dissociation for each domain should be analyzed independently and quantitatively.
      Here, we directly observed the binding and dissociation of the full-length TrCel6A (Intact) and the CD, CBM and linker (CBM-linker), and CBM of TrCel6A (Fig. 1) on crystalline cellulose Iα using single-molecule fluorescence imaging. The kon and koff values were quantitatively estimated and compared to reveal the contribution of each domain to the binding and dissociation events. Furthermore, we directly verified the processive movement of the full-length TrCel6A on crystalline cellulose Iα using single-molecule fluorescence imaging with improved localization precision (
      • Yildiz A.
      • Forkey J.N.
      • McKinney S.A.
      • Ha T.
      • Goldman Y.E.
      • Selvin P.R.
      Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.
      ) and appropriate control experiments including an catalytically inactive mutant. The ktr, duration of the movement, and the processivity of TrCel6A were estimated quantitatively at the single-molecule level for the first time.
      Figure thumbnail gr1
      FIGURE 1.Domain structures of Intact (left), CD (center left), CBM-linker (center right), and CBM (right) of TrCel6A. Pink spheres represent CD, blue spheres represent the linker, and green spheres represent CBM. Yellow spheres in CBM represent added free cysteine at position 43. Yellow spheres in CD represent the S386C mutation, and yellow spheres in the linker represent the S83C mutation. Orange spheres represent estimated sugar modifications on the linker.

      Discussion

      TrCel6A, together with TrCel7A, is one of the most well studied cellulases, and the first cellulase for which the x-ray crystal structure was determined (
      • Rouvinen J.
      • Bergfors T.
      • Teeri T.
      • Knowles J.K.
      • Jones T.A.
      Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei.
      ). In addition, the processive reaction of CBH was first proposed based on the comparison between the tunnel-like structure of TrCel6A and cleft-like structure of an endo-glucanase Thermononospora fusca Cel6A (
      • Davies G.
      • Henrissat B.
      Structures and mechanisms of glycosyl hydrolases.
      ). However, the processive movement of TrCel6A has not been directly observed, and the reaction cycle of TrCel6A was still unclear. Moreover, the role of each domain was not clear because quantitative measurements of the binding and dissociation rate constants are difficult with biochemical assays. In this report, we verified the processive movement of TrCel6A and clarified the kinetic role of the CBM and glycosylated linker region using single-molecule fluorescence imaging.
      The role of the CBM and linker region was clearly shown by the comparison of the kon and koff values among Intact, CD, and CBM-linker (Table 2). Focusing on the binding event, the CBM-linker is highly important because the kon for the CD (5.2 × 107 m−1 μm−1 s−1) was less than one-tenth that of Intact (7.5 × 108 m−1 μm−1 s−1) (Fig. 4). However, the kon for the CBM-linker was only half (3.9 × 108 m−1 μm−1 s−1) that of Intact. This difference suggests that half of the binding events of Intact are caused by the CBM-linker and half are caused by the synergistic effect of the CBM-linker and CD. Weak and transient bindings of the CBM-linker, which could not be detected as clear fluorescence signals in our observations, may help binding of the CD and increase the kon for Intact. For dissociation, both the kofffast (1.1 s−1) and koffslow (0.10 s−1) for Intact were the lowest among Intact, CD, and CBM-linker (Fig. 5). If the CBM-linker and CD of the intact molecule cannot bind to cellulose simultaneously, the koff for Intact should be same as that for the CD or CBM-linker. Therefore, the low koff values for Intact indicate that both the CD and CBM-linker simultaneously interact with the crystalline cellulose surface. Comparing the kon and koff values between Intact and the CD (with and without CBM-linker), CBM-linker caused the kon to increase 14 times and the koff to decrease to two-thirds that of the CD. Thus, the CBM-linker mainly contributes to the binding of TrCel6A by increasing the kon directly and assisting the binding of the CD. The binding of the glycosylated linker region to the cellulose surface has been demonstrated using molecular dynamics simulation (
      • Payne C.M.
      • Resch M.G.
      • Chen L.
      • Crowley M.F.
      • Himmel M.E.
      • Taylor 2nd, L.E.
      • Sandgren M.
      • Ståhlberg J.
      • Stals I.
      • Tan Z.
      • Beckham G.T.
      Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose.
      ). The synergistic binding of the CD and CBM-linker is reasonable because the freedom of the orientation of the CD will be restricted after the binding of the CBM-linker.
      After comparing kon and koff between the CBM and CBM-linker, the contribution of the glycosylated linker region becomes clear. The kon of the CBM was one-seventh that of the CBM-linker. Thus, the linker region is more important than the CBM for the initial interaction with crystalline cellulose. The main role of the glycosylation has been thought to protect cellulase from proteolysis, and information on the effect of glycosylation on the binding to crystalline cellulose is limited, especially for TrCel6A (
      • Beckham G.T.
      • Dai Z.
      • Matthews J.F.
      • Momany M.
      • Payne C.M.
      • Adney W.S.
      • Baker S.E.
      • Himmel M.E.
      Harnessing glycosylation to improve cellulase activity.
      ). One good example is the comparison of WT TrCel7A and recombinant TrCel7A produced by Aspergillus niger (
      • Jeoh T.
      • Michener W.
      • Himmel M.E.
      • Decker S.R.
      • Adney W.S.
      Implications of cellobiohydrolase glycosylation for use in biomass conversion.
      ). The mass of recombinant TrCel7A is only few hundred Daltons larger compared with the WT because of more glycosylation sites in the linker and CBM, but it binds more than twice as much as the WT. Moreover, the association constants of the CBM and CBM-linker of TrCel7A were compared, and the affinity of the CBM was reported to be only one-tenth that of the CBM-linker (
      • Payne C.M.
      • Resch M.G.
      • Chen L.
      • Crowley M.F.
      • Himmel M.E.
      • Taylor 2nd, L.E.
      • Sandgren M.
      • Ståhlberg J.
      • Stals I.
      • Tan Z.
      • Beckham G.T.
      Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose.
      ). Taken together, our results strongly suggest that the interaction between glycans on the linker region of TrCel6A and the crystalline cellulose surface causes the initial binding events. However, the koff values for the CBM were smaller than those for the CBM-linker, indicating that the CBM-linker binds crystalline cellulose mainly by the CBM and that the linker region somehow disturbs the binding of the CBM. Considering the binding mechanism of type A CBMs such as CBM1 and CBM2a with a flat hydrophobic surface, binding of the CBM-linker should be more entropically unfavorable than the isolated CBM because the binding of the CBM-linker decreases the conformational entropy of the flexible linker region (
      • Creagh A.L.
      • Ong E.
      • Jervis E.
      • Kilburn D.G.
      • Haynes C.A.
      Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven.
      ).
      The heterogeneity of the binding modes of CBH to crystalline cellulose has long been discussed. First, bindings with high and low affinities were reported for TrCel7A and attributed to its multidomain structure (
      • Stahlberg J.
      • Johansson G.
      • Pettersson G.
      A new model for enzymatic hydrolysis of cellulose based on the two-domain structure of cellobiohydrolase I.
      ). Recently, the reason for the complicated binding isotherm was explained by the steric effect using a CBM-linker connected with red fluorescent protein (
      • Sugimoto N.
      • Igarashi K.
      • Wada M.
      • Samejima M.
      Adsorption characteristics of fungal family 1 cellulose-binding domain from Trichoderma reesei cellobiohydrolase I on crystalline cellulose: negative cooperative adsorption via a steric exclusion effect.
      ). In our previous single-molecule fluorescence imaging of intact TrCel7A, we also observed two components for the koff and attributed the slow and fast components to productive and non-productive binding, respectively (
      • Shibafuji Y.
      • Nakamura A.
      • Uchihashi T.
      • Sugimoto N.
      • Fukuda S.
      • Watanabe H.
      • Samejima M.
      • Ando T.
      • Noji H.
      • Koivula A.
      • Igarashi K.
      • Iino R.
      Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.
      ). However, in the present study on TrCel6A, not only Intact and CD but also CBM-linker and CBM showed the slow and fast components (Fig. 5). In addition, two components were observed at very low sample concentrations (∼pm). These results indicate that the slow and fast components of dissociation are caused by other effects of the multidomain structure, the steric effect, and productive/non-productive bindings. The ratios between the slow and fast components for the CBM-linker and CD were 35 and 65% and 28 and 72%, respectively. If the fast component is caused by structurally unstable bindings (such as only one tryptophan residue on the CBM binds the cellulose surface), the ratio of the fast component of Intact should be smaller than that of the truncated domains. However, Intact showed ratios of 30% (slow) and 70% (fast), which were between the values for the CBM-linker and CD. Thus, this is not plausible.
      Other reasons for the two components are structural heterogeneities of cellulose. First, a single cellulose chain has polarity, i.e. reducing and non-reducing ends. Second, the ends and middle of the cellulose chain can have different interactions with CBH. Third, cellulose can form crystalline and amorphous structures. Furthermore, crystalline cellulose has flat hydrophobic and solvated hydrophilic surfaces (
      • Nishiyama Y.
      • Sugiyama J.
      • Chanzy H.
      • Langan P.
      Crystal structure and hydrogen bonding system in cellulose 1α, from synchrotron x-ray and neutron fiber diffraction.
      ). If the two bound states were caused by the binding orientation of the enzyme, the probabilities of the fast and slow components would be similar. However, all of the samples showed higher fractions of the fast component (∼70%). In addition, in our single-molecule observations, the positions at which the samples strongly bound were random and not localized to the ends or middle of the crystalline cellulose microfibrils (Fig. 9). These results rule out the first two possibilities. In this study, we used crystalline cellulose Iα prepared from Cladophora sp., and the ratio of the amorphous region was very low and less than the detection limit of XRD measurement (Fig. 10). In addition, if the surface of crystalline cellulose is highly disordered and amorphous-like, CD will show similar kon to Intact because hydrolysis activities of Intact and CD against amorphous cellulose are similar (Table 1). Therefore, the most likely reason for the two bound states is the two different surfaces of the crystalline cellulose. The CBM has a flat surface with hydrophobic, aromatic amino acid residues. It has been reported that the CBM preferentially binds to the hydrophobic surface of crystalline cellulose (
      • Boraston A.B.
      • Bolam D.N.
      • Gilbert H.J.
      • Davies G.J.
      Carbohydrate-binding modules: fine-tuning polysaccharide recognition.
      ), although the area ratio of the hydrophobic surface is much smaller than that of the hydrophilic surface in crystalline cellulose Iα (
      • Beckham G.T.
      • Dai Z.
      • Matthews J.F.
      • Momany M.
      • Payne C.M.
      • Adney W.S.
      • Baker S.E.
      • Himmel M.E.
      Harnessing glycosylation to improve cellulase activity.
      ). Thus, it is more likely that the slow and fast components correspond to the binding to the hydrophobic (high affinity) and hydrophilic (low affinity) surfaces, respectively. The dissociation constants (Kd = koff/kon) for the two binding modes are summarized in Table 3. The kon for short and long bindings were calculated from the ratio of the fast and slow components of the koff. The affinity of Intact to the hydrophobic surface was 25 and 11 times higher than that of the CD and CBM, respectively, and that to the hydrophilic surface was 19 and 28 times higher, respectively. In a previous report, the partition coefficients of the intact TrCel6A, CD, and CBM to bacterial microcrystalline cellulose were compared at nanomolar order enzyme concentrations, and the values for the intact form were 34 and 3.4 times higher than those for the CD and CBM (
      • Palonen H.
      • Tenkanen M.
      • Linder M.
      Dynamic interaction of Trichoderma reesei cellobiohydrolases Ce16A and Ce17A and cellulose at equilibrium and during hydrolysis.
      ). Thus, our results are similar to those from this previous study, although the CBM showed much lower affinity than the Intact in our study.
      Figure thumbnail gr9
      FIGURE 9.Binding specificity and distribution of Intact, Inactive, CD, CBM-linker, and CBM to crystalline cellulose. Bright field images of cellulose microfibrils (top). Single-molecule fluorescence images of each sample (middle). Fluorescence images of each sample obtained by accumulating 150 consecutive images (bottom). The scale bars are 3 μm.
      Figure thumbnail gr10
      FIGURE 10.XRD profile of crystalline cellulose Iα prepared from Cladophora sp. used in this study. Voltage and current were 40 kV and 40 mA. Scattering from the glass holder was subtracted.
      TABLE 3Values of Kd calculated from kon and koff
      Samplekon
      The kon for short and long bindings was calculated from the ratio of the fast and slow components of the koff.
      koffKd
      The unit of μm represents the length of a crystalline cellulose microfibril.
      m−1 μm−1 s−1s−1m μm
      Intact
      Low affinity site5.3 × 1081.10.21 × 10−8
      High affinity site2.3 × 1080.100.044 × 10−8
      CD
      Low affinity site3.7 × 1071.54.0 × 10−8
      High affinity site1.5 × 1070.161.1 × 10−8
      CBM-linker
      Low affinity site2.5 × 1082.61.0 × 10−8
      High affinity site1.4 × 1080.140.10 × 10−8
      CBM
      Low affinity site3.9 × 1072.35.9 × 10−8
      High affinity site1.7 × 1070.0830.49 × 10−8
      a The kon for short and long bindings was calculated from the ratio of the fast and slow components of the koff.
      b The unit of μm represents the length of a crystalline cellulose microfibril.
      Between the hydrophobic and hydrophilic surfaces, the CBM and CBM-linker showed 12 and 10 times higher affinities to the hydrophobic surface than to the hydrophilic surface, respectively. Therefore, the CBM is necessary to determine the surface specificity of binding, as shown by a molecular dynamics simulation of the CBM of TrCel7A (
      • Nimlos M.R.
      • Beckham G.T.
      • Matthews J.F.
      • Bu L.
      • Himmel M.E.
      • Crowley M.F.
      Binding preferences, surface attachment, diffusivity, and orientation of a family 1 carbohydrate-binding module on cellulose.
      ). The glycosylated linker region increases the kon and affinity to both surfaces but decreases the binding specificity to the hydrophobic surface (Table 3). It has been reported that TrCel7A hydrolyzes Valonia crystalline cellulose from the hydrophobic surface (
      • Liu Y.-S.
      • Baker J.O.
      • Zeng Y.
      • Himmel M.E.
      • Haas T.
      • Ding S.-Y.
      Cellobiohydrolase hydrolyzes crystalline cellulose on hydrophobic faces.
      ). The cellulose polymer chains accessible to the catalytic site of CBH exist mainly on the hydrophobic surface, because the cellulose chains forming the hydrophilic surface stack each other (
      • Beckham G.T.
      • Matthews J.F.
      • Peters B.
      • Bomble Y.J.
      • Himmel M.E.
      • Crowley M.F.
      Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.
      ). Thus, our result can explain the increase in non-productive binding of the recombinant TrCel7A with extra O-glycosylation in the linker and CBM (
      • Jeoh T.
      • Michener W.
      • Himmel M.E.
      • Decker S.R.
      • Adney W.S.
      Implications of cellobiohydrolase glycosylation for use in biomass conversion.
      ). Furthermore, the affinity of the CD to the hydrophobic surface is only 3.6 times higher than to the hydrophilic surface. This result suggests that the CBM leads the CD to the hydrophobic surface and increases the probability of catching the hydrolysable chain of cellulose.
      One of the most important issues regarding TrCel6A is whether it actually undergoes processive movement. With single-molecule fluorescence imaging with improved localization precision, we addressed this issue and verified that intact TrCel6A shows slow, processive movements. Thus, TrCel6A can act as a linear molecular motor moving on crystalline cellulose. The estimated value of the ktr (8.8 ± 5.5 nm/s) for TrCel6A was comparable with or slightly higher than those for TrCel7A (5–7 nm/s) determined by HS-AFM observations (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ,
      • Shibafuji Y.
      • Nakamura A.
      • Uchihashi T.
      • Sugimoto N.
      • Fukuda S.
      • Watanabe H.
      • Samejima M.
      • Ando T.
      • Noji H.
      • Koivula A.
      • Igarashi K.
      • Iino R.
      Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.
      ). Because TrCel6A has a shorter catalytic tunnel structure compared with that of TrCel7A, TrCel6A would be more easily detached from crystalline cellulose by the tapping force of the cantilever, preventing the movement under the HS-AFM. The distribution of the moving time for the slow, processive movement of TrCel6A can be fitted by a single exponential decay function with time constants of 7.7 s (Fig. 8). Considering the length (∼1 nm) of the product cellobiose (
      • Brady S.K.
      • Sreelatha S.
      • Feng Y.
      • Chundawat S.P.
      • Lang M.J.
      Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.
      ), the processivity of TrCel6A was calculated to be 68 ± 42 (8.8 ± 5.5 nm/s × 7.7 s × 1 nm−1) from the ktr and the time constant. This value is three times larger than that of TrCel7A estimated by HS-AFM observation (
      • Nakamura A.
      • Watanabe H.
      • Ishida T.
      • Uchihashi T.
      • Wada M.
      • Ando T.
      • Igarashi K.
      • Samejima M.
      Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.
      ). By the electron microscopy observation, the processivity of TrCel7A was expected to be higher than that of TrCel6A, because the edge of cellulose microfibril hydrolyzed by TrCel7A was more sharpened than that by TrCel6A (
      • Imai T.
      • Boisset C.
      • Samejima M.
      • Igarashi K.
      • Sugiyama J.
      Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals.
      ,
      • Chanzy H.
      • Henrissat B.
      Undirectional degradation of valonia cellulose microcrystals subjected to cellulase action.
      ). To compare the value of processivity of TrCel6A estimated in our study with that of TrCel7A quantitatively, the TrCel7A also need to be analyzed with single-molecule fluorescence imaging.
      It has been reported that fungal glycoside hydrolase 6 (GH6) enzymes has mixed modes of endo- and exo-initiations (
      • Boisset C.
      • Fraschini C.
      • Schülein M.
      • Henrissat B.
      • Chanzy H.
      Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A.
      ). In our analysis, however, we could not distinguish endo- and exo-initiations of TrCel6A. Furthermore, we cannot exclude the possibility that TrCel6A also acts non-processively, because a significant fraction of Intact showed dissociation without translational movement.
      The CD of TrCel6A has shorter loops covering the catalytic tunnel than that of a bacterial GH6 cellulase from Thermobifida fusca Cel6B (
      • Wu M.
      • Bu L.
      • Vuong T.V.
      • Wilson D.B.
      • Crowley M.F.
      • Sandgren M.
      • Ståhlberg J.
      • Beckham G.T.
      • Hansson H.
      Loop motions important to product expulsion in the Thermobifida fusca glycoside hydrolase family 6 cellobiohydrolase from structural and computational studies.
      ), and structures of the linker region and CBM are also different between fungal and bacterial GH6 cellulases. Therefore, the correlation between structure and function such as between loop length and degree of processivity is an interesting question that can be assessed by single-molecule fluorescence imaging. By quantitatively analyzing the role of each domain of not only fungal but also bacterial GH6 cellulases, valuable insights to engineer more efficient hybrid cellulases will be gained.
      Previous single-molecule studies have reported processive movements of the isolated CD of TrCel7A (
      • Igarashi K.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Penttilä M.
      • Samejima M.
      High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose.
      ,
      • Brady S.K.
      • Sreelatha S.
      • Feng Y.
      • Chundawat S.P.
      • Lang M.J.
      Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.
      ). However, in our study, we did not observe the CD of TrCel6A undergoing slow and long movement (Fig. 7). One possible explanation is that the ratio of productive binding decreased in the absence of the CBM-linker, as we described above. Alternatively, the absence of the CBM-linker may significantly decrease the processivity. Regarding this second possibility, Bu et al. (
      • 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.
      The energy landscape for the interaction of the family 1 carbohydrate-binding module and the cellulose surface is altered by hydrolyzed glycosidic bonds.
      ) predicted that the CBM has important roles in not only binding but also introducing a single cellulose chain to the catalytic site and increasing the processivity of the reaction. Their molecular simulation showed that the CBM exerts a driving force and assists processive movement by biased sliding when the CBM binds near the cellulose chain end. However, the effect of the CBM-linker on the processivity of TrCel6A is still open for discussion.
      In addition to the slow and long processive movements of Intact, fast (30–40 nm/s) and short (<10 s) movements were observed for all samples. We attributed these fast movements to diffusion on the surface of crystalline cellulose. TrCel6A may employ multiple strategies to catch the hydrolysable point of the cellulose chain efficiently: direct binding from solution and diffusional searching on the crystalline cellulose surface. In the near future, high resolution and long-term single-molecule imaging with much brighter optical probes such as quantum dots (
      • Kairdolf B.A.
      • Smith A.M.
      • Stokes T.H.
      • Wang M.D.
      • Young A.N.
      • Nie S.
      Semiconductor quantum dots for bioimaging and biodiagnostic applications.
      ) and gold nanoparticles (
      • Isojima H.
      • Iino R.
      • Niitani Y.
      • Noji H.
      • Tomishige M.
      Direct observation of intermediate states during the stepping motion of kinesin-1.
      ,
      • Ueno H.
      • Minagawa Y.
      • Hara M.
      • Rahman S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Torque generation of Enterococcus hirae V-ATPase.
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ) will be applied to TrCel6A and other CBHs. These studies will resolve the detail of the entire cycle of the processive reaction including the decrystallization, chain threading, bond cleavage, and product release and will give valuable insights for the rational design and engineering of better, non-natural cellulases.

      Author Contributions

      A. N. conducted all experiments and data analysis and wrote the manuscript. T. T., D. I., A. V., and M. M. conducted single-molecule experiments and analysis. M. Y. and Y. O. prepared samples. T. T. and T. U. conducted hydrolysis activity measurements. H. N., M. S., K. I., and R. I. organized the experiments. R. I. conceived the project and wrote the manuscript with A. N.

      Acknowledgments

      We thank Dr. Takayuki Uchihashi and Dr. Kentaro Ishii for helpful discussion and Kaori Nakane for administrative assistance.

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