DirectedAssembly of Homopentameric Cholera ToxinB-Subunit Proteins into Higher-Order Structures Using Coiled-CoilAppendages

摘要

The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein–protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are prescreened for “designability”, limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multisubunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. Analysis of a tubular structure determined by X-ray crystallography has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which presenteither termini on a single face of an oligomer. This scaffolding approachcan be used to generate bespoke supramolecular assemblies of functionalproteins. Additionally, structural resolution of the scaffolded assemblieshighlight “native-state” forced protein–proteininterfaces, which may prove useful as starting conformations for futurecomputational design.