Modeling the self-assembly of the cellulosome enzyme complex

Abstract

Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of cellulosomes created during self-assembly. Here, we present a coarse-grained model to study the self-assembly of cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multimodular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multimodularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers cellulosomes.

FIGURE 1.

Concept of a cellulosome from C. thermocellum. The scaffoldin subunit (dark blue) contains nine cohesins and a carbohydrate binding module. The cellulolytic enzymes (gray) bind to cohesin partners with their dockerins. Another set of dockerin/cohesin interaction connects the scaffoldin to cell wall via a S-layer homologous (SLH) protein.

FIGURE 2.

Sequence-based PONDR screen for protein disorder applied to CipA. a, the VL3 algorithm (58) predicts the CipA linkers to be disordered regions; these are the regions with scores greater than 0.5. b, the CipA scaffoldin colored by VL3 scores where the minimum score is 0 (blue) and the maximum VL3 score is 1.0 (red) show the predicted ordered and disordered regions of the scaffoldin. The X domain and dockerin module at the C terminus of the scaffoldin were omitted.

FIGURE 3.

Mean net charge as a function of hydropathy for the CipA linkers with an average length of 25 residues. The training sets for disordered and ordered proteins are shown in red and blue, respectively.

FIGURE 4.

Coarse-grained representation and all atom representation of CipA from C. thermocellum. The structures of the CBM and one of the cohesins are known and reported in the literature (48, 55). The other cohesins were obtained from homology modeling.

FIGURE 5.

All atom and coarse-grained representations of the cohesin from CipA with the attractive beads shown in red.

FIGURE 6.

All atom and coarse-grained representations of CelS (GH48), Cel5B (GH5), and CbhA (GH9).

FIGURE 7.

Simulation box with a scaffoldin molecule and some cellulosomal enzymes. The enzymes bound on the scaffoldin have solid colors. The color coding is the following: CelS (red), Cel5B (green), CbhA (blue).

FIGURE 8.

Summary of the binding studies conducted. Scan 1, original masses and volumes/shapes are shown. Scan 2, same volumes/shapes and different masses are shown; Scan 3, different volumes/shapes and same masses are shown.

FIGURE 9.

The ratio of the solution fraction to the 4-cohesin-scaffold-bound fraction for total enzyme concentration of 60 enzymes per box with original masses and shapes. The solid line represents an equal enzyme ratio in both solution and on the scaffoldin.

FIGURE 10.

The ratio of solution fraction to the 4-cohesin-scaffold-bound fraction for a total enzyme concentration of 60 enzymes per box with original masses and shapes for a ratio between 0 and 3. The solid line represents an equal enzyme ratio in both solution and on the scaffoldin.

FIGURE 11.

The ratio of solution fraction to the 4-cohesin-scaffold-bound fraction for a total enzyme concentration of 30 enzymes per box with original masses and shapes. The solid line represents an equal enzyme ratio in both solution and on the scaffoldin.

FIGURE 12.

The ratio of solution fraction to the 4-cohesin-scaffold-bound fraction for a total enzyme concentration of 60 enzymes per box with same volumes/shapes and different masses. The system is described in Fig. 8.

FIGURE 13.

The ratio of solution fraction to the 4-cohesin-scaffold-bound fraction for a total enzyme concentration of 60 enzymes per box with different volumes/shape and same masses. The system is described in Fig. 8.

FIGURE 14.

Residence time around the scaffoldin for Cel5B, CelS, and CbhA using three cases from Scan 1 with S.D. (1 S.D.).