Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module

Abstract

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180° rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wild-type cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

Figure 1

Schematic representation of cellulosome organization in C. thermocellum. The cellulosomal structural and enzymatic subunits comprise modular components. The cellulosomal enzymes each carry one or more catalytic modules and a single dockerin module. The scaffoldin is multifunctional, whereby the nine cohesin modules (enumerated) integrate nine dockerin-bearing enzymes into the complex, the carbohydrate-binding module (CBM) binds the complex to the cellulosic substrate, and the C-terminal dockerin module is involved in anchoring the cellulosome complex to the bacterial cell surface. The modules are separated from each other by defined linker segments.

Figure 2

The top panels show structures of the Coh-Doc complex bound in mode I (A) and II (B). Coh and Doc are shown in blue and red, respectively. The spatial orientation of Coh is the same on both panels. The structure of Doc on panel B (mode II) is rotated by 180° relative to the structure of Doc on panel (A) (mode I). The three α-helices forming the Doc domain are labeled as ${\alpha }_1\prime$, ${\alpha }_2\prime$ and ${\alpha }_3\prime$. The Doc-bound Ca²⁺ ions are shown as gray spheres. The Doc residues making contacts with the Coh in the binding mode I include R23, L22, N44, S45, T46 and R53. They are shown in the van der Waals representation. The Doc residues making contacts with the Coh in mode II include R57, L56, N10, S11, T12 and R19. The locations of R57, L56, N10, S11, T12 and R19 in mode II are analogous, respectively, to the positions of R23, L22, N44, S45, T46 and R53 in mode I. Panel (C) shows structures of the Coh-Doc complex given by the PDB:1OHZ (Coh in blue, Doc in red) and PDB:2CCL (Coh in cyan, Doc in orange). The Coh structures are superimposed. The spheres represent the dockerin-bound Ca²⁺ ions. The Doc helices are not aligned, indicating the existence of the two different binding modes. Panel D shows the PDB structure 1OHZ. Coh and Doc are shown in blue and red, respectively. The black line shows the derived axis of the symmetry, denoted here as the Z-axis. The sense of the rotation is indicated at the top.

Figure 3

Results of the FoldX-based free-energy calculations for system C_ID_I (panels A,B and C) defined in Table 1 for index k = 1, and for system $C_I{d}_I^{⁎}$ (panels D–F) corresponding to index k = 5 in Table 1. (A) ΔG as a function of coordinates Z and φ. The values of ΔG are indicated by the color scale shown on the right hand side. (B) ΔG as a function of Z for angles $\varphi =3^{\circ }$ (top sub-panel, mode I) and $\varphi ={173}^{\circ }$ (bottom sub-panel, mode II) at which $\mathrm{\Delta }G(Z,\varphi )$ is found to take the minimal values, $\mathrm{\Delta }{G}_{\mathrm{m}in}=-38.2$ kcal/mol and $\mathrm{\Delta }{G}_{\mathrm{m}in}=-28.7$ kcal/mol, respectively. The data points in cyan show the results of the FoldX-based calculations. The solid blue lines correspond to a harmonic approximation at the minimum of ΔG(Z). (C) ΔG minimized with respect to Z and plotted as a function of φ. The data points in cyan show the results of the FoldX-based calculations. The solid blue line represents a fit to the data points. This line is a to guide the eye. The fitting function used here involves three Gaussians. Panels (D–F) are analogous to panels (A–C), respectively.

Figure 4

Free energies of binding in mode I (red) and II (green) as functions of the energy cut-off, E_c, which is used to compute F_I and F_II according to Eqs (1) and (2). The top panels, A and B, correspond to the Doc without the tails (sequences comprising residues 1 through 56). The bottom panels, (C and D), correspond to the full-length Doc (sequences comprising residues −5 through 56). Four cases are considered: (A) Coh-Doc in which Doc is WT and without the tails. Here, F_I and F_II are computed by taking $k=\mathrm{1,}\dots \mathrm{,4}$. (B) Coh-Doc^* in which Doc^* is the two-point mutant without the tails. The binding energies are computed by taking $k=\mathrm{5,}\dots \mathrm{,8}$ in this case. (C) Coh-Doc in which Doc is WT and full-length, i.e., comprising residues −5 through 56. This case corresponds to averaging over $k=\mathrm{9,}\dots \mathrm{,14}$ in Eqs (1) and (2). (D) Coh-Doc^* in which Doc^* is the two-point mutant with the tails, i.e., comprising residues −5 through 56. Structural models with $k=\mathrm{15,}\dots \mathrm{,20}$ have been used for calculating F_I and F_II in this case.

Figure 5

Analogous to Fig. 3 but for system C_IID_II (panels A–C) corresponding to index k = 10 in Table 2, and for system $C_{II}{D}_{II}^{⁎}$ (panels D–F) corresponding to index k = 16 in Table 2.

Figure 6

Force-displacement curves for the WT system C_ID_I with k = 9 (left panels) and for the mutated system $C_{II}{D}_{II}^{⁎}$ with k = 19 (right panels). The upper panels show typical long trajectories. The lower panels correspond to short trajectories. The symbols indicate the contacts that break at particular displacements. The primed symbols refer to Doc and the unprimed to Coh. N’ is the region near the N-terminus in Doc. For the short trajectory in the lower panel, the contacts ${\alpha }_1\prime -{\beta }_{\mathrm{3,6,8}},{\alpha }_3\prime$ break both around 130 and 140 Å, i.e., at the two last force peaks.