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

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 I_α 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.

Keywords: cellulase; cellulose; enzyme kinetics; enzyme mechanism; microscopic imaging; molecular motor; processivity; protein domain; single-molecule biophysics.

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.

FIGURE 2.

A, domain constructions and mutated amino acid residues of the samples. B, SDS-PAGE of the samples used in this study. Lane 1, WT; lane 2, Intact; lane 3, Inactive; lane 4, CD; lane 5, CBM-linker; lane 6, CBM. Samples (10 pmol) were loaded in lanes 1–4, and 30 pmol and 100 pmol of sample was loaded in lanes 5 and 6, respectively. Samples were separated in a 15–20% gradient gel with Tris-Tricine buffer.

FIGURE 3.

Hydrolysis activities of WT and Intact at various substrate concentrations. Hydrolysis activities of WT (black circles) and Intact (yellow triangles) at various substrate concentrations (from 0.03 to 0.5% (w/v)) and the Michaelis-Menten fit are shown. k_cat values of 3.1 s⁻¹ and 2.8 s⁻¹ and K_m values of 0.26% (w/v) and 0.27% (w/v) were obtained for WT and Intact, respectively.

FIGURE 4.

Distributions of the binding rate constant (k_on) for Intact (left), CD (center left), CBM-linker (center right), and CBM (right). The distributions of Intact, CD, CBM-linker, and CBM were fitted with two or three Gaussians. The concentrations of the samples were 25, 250, 50, and 200 pm for Intact, CD, CBM-linker, and CBM, respectively. The number of cellulose microfibrils analyzed was 170, 106, 104, and 193 for Intact, CD, CBM-linker, and CBM, respectively. The bin widths were 1.25 × 10⁸ m⁻¹ μm⁻¹ s⁻¹ for Intact and CBM-linker, 1.4 × 10⁷ m⁻¹ μm⁻¹ s⁻¹ for CD, and 2.0 × 10⁷ m⁻¹ μm⁻¹ s⁻¹ for CBM.

FIGURE 5.

Distributions of the duration time on cellulose for Intact (left), CD (center left), CBM-linker (center right), and CBM (right) fitted with double exponential decay functions. For Intact, k_off^fast was 1.1 s⁻¹ (70%), and k_off^slow was 0.10 s⁻¹ (30%). For CD, k_off^fast was 1.5 s⁻¹ (72%), and k_off^slow was 0.16 s⁻¹ (28%). For CBM-linker, k_off^fast was 2.6 s⁻¹ (65%), and k_off^slow was 0.14 s⁻¹ (35%). For CBM, k_off^fast was 2.3 s⁻¹ (70%), and k_off^slow was 0.083 s⁻¹ (30%). The number of events was 757, 403, 614, and 756 for Intact, CD, CBM-linker, and CBM, respectively. The bin widths of the distributions were 0.2 s.

FIGURE 6.

Trajectories of the movement for Intact, Inactive, CD, and CBM-linker. The red plots show the centroid trajectory of the movement. The blue plots show the centroid trajectory of a non-moving molecule in the same field of view and demonstrate that the coverslip did not drift. The frame rate of observation was 0.5 fps. The scale bars are 40 nm for both the vertical and horizontal axes.

FIGURE 7.

Distribution of translational rate constant (k_tr) for Intact, Inactive, CD, and CBM-linker and plots of k_trversus moving time on crystalline cellulose. Distribution of the k_tr for Intact was fitted by two Gaussians; the peak ± S.D. values were 8.8 ± 5.5 and 34.9 ± 16.3 nm/s (n = 180), respectively. Distributions of the k_tr for Inactive, CD, and CBM-linker were fitted by a single Gaussian. The peak ± S.D. values were 35.5 ± 17.4 nm/s (n = 174), 39.2 ± 28.3 nm/s (n = 102), and 42.6 ± 28.0 nm/s (n = 105), respectively.

FIGURE 8.

Distribution of moving time for Intact showing slow (0–20 nm/s), processive movement. Molecules that moved for longer than 10 s are highlighted in pink. The distributions were fitted by single exponential decay functions: y = 31.9 × exp(−0.13 × t) (all range) and y = 36.4 × exp(−0.13 × t) (>10 s). The bin width was 2 s. The numbers of molecules were 100 (all range) and 40 (>10 s).

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 10.

XRD profile of crystalline cellulose I_α prepared from Cladophora sp. used in this study. Voltage and current were 40 kV and 40 mA. Scattering from the glass holder was subtracted.