Abstract
Cellulose, the main structural component of plant cell walls, is the most abundant carbohydrate polymer in nature. To break down plant cell walls, anaerobic microorganisms have evolved a large extracellular enzyme complex termed cellulosome. This megadalton catalytic machinery organizes an enzymatic assembly, tenaciously bound to a scaffolding protein via specialized intermodular "cohesin-dockerin" interactions that serve to enhance synergistic activity among the different catalytic subunits. Here, we report the solution structure properties of cellulosome-like assemblies analyzed by small angle x-ray scattering and molecular dynamics. The atomic models, generated by our strategy for the free chimeric scaffoldin and for binary and ternary complexes, reveal the existence of various conformations due to intrinsic structural flexibility with no, or only coincidental, inter-cohesin interactions. These results provide primary evidence concerning the mechanisms by which these protein assemblies attain their remarkable synergy. The data suggest that the motional freedom of the scaffoldin allows precise positioning of the complexed enzymes according to the topography of the substrate, whereas short-scale motions permitted by residual flexibility of the enzyme linkers allow "fine-tuning" of individual catalytic domains.
Highlights
Cellulolytic bacteria living in anaerobic biotopes produce large extracellular multienzymatic complexes termed cellulosomes that efficiently degrade crystalline cellulose and related plant cell wall polymers [1]
The simplest cellulosomes, such as those produced by Clostridium cellulolyticum, include eight enzymes tenaciously bound to a scaffolding protein bearing a cellulose-binding module (CBM)3 that anchors the entire
Such artificial assemblies were designed to comprise a chimeric scaffoldin, possessing an optional CBM and two cohesins of divergent specificity and two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. In these artificial cellulosomes, the composition of enzymes and their position on the hybrid scaffoldin can be strictly controlled. The design of these chimeric components allowed the investigation of an apparent paradox: the action of cellulosomes on crystalline cellulose is catalytically efficient, whereas their individual enzymatic components display relatively low activity and one cohesin from C. cellulolyticum connected by a linker peptide of 51 residues; Fc-S4, S4 in complex with one native full-length Cel48F from C. cellulolyticum; Fc-S4Ft, S4 in complex with two identical cellulases Cel48F appended with either a C. cellulolyticum or a C. thermocellum dockerin; Fc-S4-At, S4 in complex with two different cellulases, namely Cel48F appended with a C. cellulolyticum dockerin and cellulase Cel5A appended with a C. thermocellum dockerin
Summary
Preparation of Protein Samples for SAXS—The production and purification of the different components (the chimeric scaffoldin S4 that contains a C. cellulolyticum cohesin fused to a C. thermocellum cohesin, the C. cellulolyticum cellulase Cel48F appended with either its native C. cellulolyticum dockerin or a C. thermocellum dockerin, and Cel5A appended with a C. thermocellum dockerin) were described previously [5]. The program package CREDO [14] was used to add missing domains (from 250 residues for S4 to 910 residues for Fc-S4-Ft) by fixing the known atomic structures and building the unknown regions to fit the experimental scattering data obtained from the entire particle. This procedure was applied in all cases to restore the low resolution shapes of missing linker regions. For each registered conformation the theoretical SAXS profile, the RG, and the corresponding fit to the experimental data were calculated using the program CRYSOL [19]
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