Browsing by Author "Cameron, Kate"
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- Small angle X-ray scattering analysis of Clostridium thermocellum cellulosome N-terminal complexes reveals a highly dynamic structurePublication . Currie, Mark A.; Cameron, Kate; Dias, Fernando M. V.; Spencer, Holly L.; Bayer, Edward A.; Fontes, Carlos M. G. A.; Smith, Steven P.; Jia, ZongchaoClostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.
- Structure and function relationships in novel cohesin-dockerin complexesPublication . Cameron, Kate; Fontes, Carlos Mendes Godinho de Andrade; Najmudin, ShabirCohesin-dockerin interactions orchestrate the assembly of carbohydrate degrading multi-enzyme complexes produced by anaerobic bacteria termed cellulosomes. Type I dockerins typically display a dual binding mode which has been suggested to allow increased flexibility for cellulosome assembly. In contrast, structural work on type II dockerins suggests that they display a single binding mode. In this work structure function studies were developed in the cellulosomal systems of Clostridium thermocellum, Bacteroides cellulosolvens, and Acetivibrio cellulolyticus. The data provides novel structural and dynamic insights into the mechanism of substrate recognition by cellulosomes (Chapter 2). In addition, to understand the mechanism of cellulosome assembly in more elaborate cellulosomal systems, structural studies of novel type I and type II cohesin-dockerin complexes of B. cellulosolvens and A. cellulolyticus were developed. The crystal structure of a type I cohesin from B. cellulosolvens cell surface anchoring scaffoldin ScaB is reported (Chapter 3). This type I cohesin is highly similar to the type I cohesins from C. thermocellum and C. cellulolyticum and its cognate type I dockerin displays a dual binding mode. In Chapter 4, the structure of the type II X-dockerin from A. cellulolyticus in complex with a type II adaptor cohesin in two distinct orientations is described. The dockerin displays structural symmetry which is reflected by the presence of two essentially identical cohesin binding surfaces, suggesting that flexibility modulated by the dockerin dual binding mode is extended to type II complexes. In Chapter 5, the structure of a A. cellulolyticus type I cohesin-dockerin complex involved in cell surface attachment is described. Typical of type I cohesin-dockerin interactions this dockerin displays a dual binding mode, with a complex interface much more extensive than that observed in other type I complexes, resulting in an extremely tight interaction (Ka ~ 1012 M). Furthermore, data reveal that residues located at dockerin positions 12, 14 and 19 modulate the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. In conclusion, this work demonstrates the importance of the dockerin dual binding mode to incorporate additional flexibility to cellulosome assembly and also polycellulosome assembly and cell surface attachment.