Structural and functional investigation of the cytoplasmic domain of the Fas death receptor

Activation of the transmembrane death receptor Fas (CD95/APO-1) by a membrane bound ligand (FasL/CD95L) activates the extrinsic pathway of apoptosis. Intracellular Fas death domains (DDs) are induced to oligomerise enabling binding to the adaptor protein FADD, thereby leading to the recruitment of procaspase 8 and other proteins to form the death inducing signalling complex (DISC).This thesis describes an investigation of the structure and function of the cytoplasmic Fas-DD. A model for the solution structure of the Fas-DD was published in 1996, it has since been reported that the death domain can form at least one other conformation when in complex with FADD. As a foundation to the work in this thesis, modern multidimensional NMR techniques have been used to solve the structure of the FasDD, to further probe the potential for alternative conformations. It has previously been reported that Fas can be phosphorylated at Tyr291, providing a platform for the recruitment of binding partners that can affect non-apoptotic signalling. The second part of this thesis details the development of an expressed protein ligation methodology to prepare a Tyr291 phosphorylated Fas DD to provide a basis for in vitro studies of the structural, dynamic and functional effects of phosphorylation. It is widely accepted that Fas is palmitoylated at Cys199 and recognised by the membrane cytoskeletal protein, ezrin. Fas palmitoylation is important for clathrinmediated internalisation of the DISC, and amplification of the caspase cascade. There are multiple reports detailing the binding of ezrin to Fas, but it is not clear whether this interaction occurs in a palmitoylation-dependent manner. Efforts to characterise an interaction between bacterially expressed intracellular Fas and ezrin proteins were carried out using a number of biophysical assays, described in the third part of this thesis. Building upon this, the fourth section explores the preparation of a palmitoylated Fas construct suitable for biophysical analysis by incubating recombinant Fas with palmitoyl-CoA

Directed Assembly of Homopentameric Cholera Toxin B‑Subunit Proteins into Higher-Order Structures Using Coiled-Coil Appendages

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 pre-screened for ‘designability’, limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multi-subunit 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. X-ray crystallography and analysis of a tubular structure 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 present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight ‘native-state’ forced protein-protein interfaces, which may prove useful as starting conformations for future computational design

Membrane Fusion Mediated by Non-covalent Binding of Re-engineered Cholera Toxin Assemblies to Glycolipids

Membrane fusion is essential for the transport of macromolecules and viruses across membranes. While glycan-binding proteins (lectins) often initiate cellular adhesion, subsequent fusion events require additional protein machinery. No mechanism for membrane fusion arising from simply a protein binding to membrane glycolipids has been described thus far. Herein, we report that a biotinylated protein derived from cholera toxin becomes a fusogenic lectin upon cross-linking with streptavidin. This novel reengineered protein brings about hemifusion and fusion of vesicles as demonstrated by mixing of fluorescently labeled lipids between vesicles as well as content mixing of liposomes filled with fluorescently labeled dextran. Exclusion of the complex at vesicle–vesicle interfaces could also be observed, indicating the formation of hemifusion diaphragms. Discovery of this fusogenic lectin complex demonstrates that new emergent properties can arise from simple changes in protein architecture and provides insights into new mechanisms of lipid-driven fusion