Understanding Cellulosome Interaction with Cellulose by High-Resolution Imaging.

Molecular observations of an enzyme reacting on its substrate is of prime interest to understand the correlation between protein function and its structural dynamics. The study by Eibinger and colleagues,[1] in this issue of ACS Central Science, uses atomic force microscopy (AFM) to capture high-resolution snapshots, in real time at the minute time scale, of enzyme reaction on a solid substrate at near-physiological conditions. This study demonstrated the structural changes of the cellulosome, a protein complex, as it binds to and reacts on a cellulosic substrate.

Imaging techniques have expedited cellulosome discovery and development in the past few decades. In the very earliest studies that documented the discovery of the cellulosome,[2] negative staining electron microscopy played a prominent role in revealing high-molecular-weight protein particles that contain multiple subunits and can be isolated from growth cultures of the cellulose-degrading bacterium Clostridium thermocellum. Further studies showed that cellulosomes contain repeated structures that bind to cellulose and completely hydrolyze it.[3] Therefore, cellulosomes were described as multienzyme complexes.[2,4] Molecular biology and genetic studies have further demonstrated that these multiple protein complexes are composed of a variety of enzymes, each bearing a single dockerin module and at least one nonenzymatic “scaffolding” component that contains multiple cohesin modules.[4] The specific interaction between the dockerin and cohesin modules dictates the assembly of numerous enzymes onto the primary scaffolding subunit that, in turn, anchors onto the bacterial cell surface via a divergent type of cohesin–dockerin interaction. Thus, the cellulosome represents a unique enzymatic system existing in selected species of Clostridia and other related bacteria, which demonstrates superior activities in degrading complex cellulosic substrates, such as plant cell walls (Figure ). Broad mechanistic understanding of the dynamic structure of these protein complexes will support advanced biotechnological and nanotechnological engineering of novel types of cellulosome-based complexes. This would, for example, help to develop effective enzyme systems for biomass conversion that would enable economic production of lignocellulosic biofuels and biomaterials.[4]

Figure 1

Schematic representation of cellulosome structure and its functional interaction with the major plant cell wall-degrading components, i.e., cellulose, hemicellulose, and lignin. As an example, the cellulosome system of the cellulolytic bacterium C. thermocellum is shown. The cellulosome complex binds to the cellulose fibrils in the plant cell walls via the cellulose-binding module of the CipA primary scaffold protein and is tethered to the bacterial cell wall via the surface-layer homology module of the OlpA anchoring scaffold protein. The type-I dockerin-bearing enzymes bind to the nine type-I cohesins of the primary CipA scaffold protein, whose type-II dockerin binds to the seven type-II cohesins of the OlpB anchoring protein. Thus, a single cellulosome assembly on the C. thermocellum surface can contain 63 different types of enzymes, including cellulases, hemicellulases, and other glycoside hydrolases that work synergistically to degrade the different plant cell wall polysaccharides.

In the 1980s, imaging studies were instrumental in the discovery of the cellulosome. Following two decades of subsequent research, scientists proposed that the scaffold protein is highly flexible for integrating multiple enzymes into a complex, thereby promoting synergistic activity as a function of their structural proximity. Precise organization and assembly of cellulosomes on the bacterial cell surface was recently documented by using advanced imaging approaches, that is, super-resolution or stochastic optical reconstruction microscopy (STORM) and correlative light and electron microscopy (CLEM) (see Figure A).[5] Crystallographic evidence, small-angle X-ray scattering (SAXS) techniques,[6,7] and biochemical studies of recombinant cellulosomal components have indeed confirmed that the scaffold protein exhibits considerable freedom of motion and that its intermodular linker segments facilitate flexible accessibility of the catalytic domains to the substrate.

Figure 2

Imaging cellulosome organization on the bacterial cell surface and how it binds to and reacts with plant cell wall cellulose. (A) Three-dimensional super-resolution image of fluorescently labeled cellulosomal enzymes (green) and scaffold protein (red), bound to the surface of Clostridium clariflavum.[5] (B, C). Confocal laser scanning microscopy of plant cell walls from corn stover, showing binding of fluorescently labeled free (uncomplexed) fungal cellulases, penetrating into the cell wall (B), and cellulosomes bound to, but not penetrating, the wall surface (C).[9] Scalebar: 2 μm (A), 5 μm (B, C).

Atomic force microscopy (AFM) offers a unique tool to observe biomolecules at molecular resolution under near-physiological conditions. These studies provide real-time observation of cellulase binding to cellulose and in situ morphological changes of the substrate during enzyme reaction. At the minute time scale, AFM imaging revealed different mechanisms of action between free (uncomplexed) fungal cellulases versus bacterial cellulosome complexes, during the process of hydrolyzing cellulosic substrates. Reactions of free fungal cellulases on crystalline cellulose substrates were first imaged by AFM,[8] where individual cellulases were shown to “run” processively along the hydrophobic surface of the cellulose crystal. Subsequently,[9] bacterial cellulosome complexes were imaged by AFM on pretreated plant cell walls that contain lignin and hemicellulose in addition to cellulose. In the latter work, the cellulosomes remained localized on the cellulosic substrate[9,10] in a “digging”-type mode,[1] as opposed to the “running” mode exhibited by the free fungal cellulases.[8] Thus, in a complex biomass like the plant cell wall, the relatively small fungal cellulases can penetrate into the fibrillar matrix of cell walls, whereas cellulosomes remain primarily on the surface due to their large size (Figure B,C).[9]

Although early electron microscopy observations have demonstrated the dynamic conformational changes of cellulosomes from tightly packed particles to loosely elongated shapes upon binding to the cellulose surface,[2,3] later studies primarily focused on structural changes of the cellulosic substrates.[2,10] The current research[1] further demonstrates the direct imaging of these dynamic protein assemblies with video recording, thereby highlighting, experimentally and in real time, the conformational changes of cellulosomes while binding to and digesting plant-derived cellulose nanocrystals.