The cohesin module is a major determinant of cellulosome mechanical stability

Abstract

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.

Keywords: atomic force microscopy (AFM); cellulosome; dockerin; linkers; nanomechanics; protein stability; scaffold protein; scaffoldin; single-molecule biophysics; single-molecule force spectroscopy.

Figure 1.

Cohesins on the connecting region of primary scaffoldins show high mechanical stability. A, representation of C. thermocellum scaffoldin CipA; the modules studied in this work are highlighted. The inset describes the symbols used. Also shown are unfolding force (left) and ΔL_C (right) histograms for CtA1 (green, n = 70) and CtA9 (blue, n = 59). A representative force extension recording of each protein is plotted at the right. A schematic of the protein used for AFM-SMFS analysis is shown at the right of the recordings; five I27 repeats are used as single-molecule markers (black in the force extension traces). B, schematic of A. cellulolyticus ScaA scaffoldin. Also shown are unfolding force (left) and ΔL_C (right) histograms of AcA3 (yellow, n = 187) and AcA4 (red, n = 37). C, dynamic force spectra of CtA1, CtA9, and the single-molecule marker I27 (black). Open symbols represent unfolding forces calculated using Monte Carlo simulations. Histograms in A and B show normalized frequencies.

Figure 2.

Mechanical properties of representative cohesins from secondary scaffoldins of A. cellulolyticus. A, representation of two A. cellulolyticus secondary scaffoldins (ScaB and ScaC); the selected cohesins studied are highlighted and enumerated. The inset describes the symbols used. B and C, unfolding force (B) and ΔL_C (C) histograms of AcB4 (yellow, n = 59) and AcC3 (blue, n = 58) fitted to Gaussian curves. D, schematic of the constructions used for this analysis and force extension recordings of their unfolding. Cohesin unfolding is highlighted in the corresponding color, whereas black peaks show the unfolding of the single-molecule markers. Above the recordings, schematics of constructions used are shown, similar to Fig. 1A. Histograms in B and C show normalized frequencies.

Figure 3.

The intermodular linkers and the presence of multiple cohesins do not affect the mechanical stability of cohesin modules. A, schematic of C. cellulolyticum CipC scaffoldin. The modules studied are highlighted, and lines indicate the fragment of the scaffoldin included in each analysis. B and C, unfolding force (left) and ΔL_C (right) histograms of CcC1Link (n = 105, B), CcC123 (n = 586, C) and CcC3 (n = 110). A schematic of the construction used in each case is also shown. The data for CcC1 and CcC1–7 correspond to those reported in Ref. , which are plotted here for comparison. Histograms in B and C show normalized frequencies.

Figure 4.

Dockerin binding does not affect the mechanical stability of cohesins. A, schematic of the C. thermocellum CipA scaffoldin; CtA2 (yellow) and CtA7 (red) are highlighted. B–D, unfolding force (B) and ΔL_C histograms of CtA2 (C) and CtA7 (D) in the absence (top, n = 69 and n = 102, respectively) or presence (bottom, n = 185 for CtA2 and n = 40 for CtA7) of Cel8A (a C. thermocellum enzyme containing the appropriate dockerin for interaction with the test cohesin). E–G, superposition of 1OHZ (E), 2CCL (F), and CtA7-Dockerin model (G) force extension traces calculated using SMD. Red triangles indicate the first frame of the trajectory where cohesin–dockerin interaction was lost. The structure of each complex is shown above the trajectories; the N terminus of each component is marked with a blue sphere. Histograms in B, C, and D show normalized frequencies.

Figure 5.

Refolding kinetics of cohesin modules. A, simultaneous extension-time (top) and force-time (bottom) traces for CtA1 (red) and CtA7 (blue). For CtA1, a 90 pN for 2 s force pulse was applied to unfold the protein (shown in higher magnification in the inset), whereas a ramp from 150 to 550 pN in 2s was used to unfold CtA7. Then force was reduced during the relaxation time and a second unfolding step was applied to probe the number of refolded modules. B, fraction of refolded modules as a function of the relaxation time.