Nematollahi, LA; (2013) NMR and Biophysical Investigation of Death Domain Assemblies. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Homotypic death domain (DD)-death domain interactions are important in the assembly of oligomeric signalling complexes such as the CD95/Fas DISC, PIDDosome, and the RIPoptosome. These complexes are considered as platforms for triggering apoptotic and other downstream signalling pathways. The first part of this thesis is aimed at characterisation of the PIDDosome core complex in solution using different techniques, with a specific focus on the application of methyl-TROSY NMR experiments. Analysis of the stoichiometry and molecular weight (MW) of the intact complex by AUC, SEC-MALS, and Nano-ESI confirmed the formation of high MW species (130-158 kDa) with a flexible stoichiometry of 10-12 chains that is not in complete agreement with previously reported data. Standard (15N, 1H)-HSQC titration experiments displayed global loss of signal confirming the formation of high MW species. 13C-methyl-TROSY experiments of the complex showed splitting of the cross peaks and evidence of differential line broadening and chemical shift heterogeneity that reflects the presence of non-equivalent sites within the different inter-domain interfaces within the asymmetric complex. Methyl-TROSY spectra for ILV-labelled samples titrated with unlabelled binding partners were characterised by chemical shift changes in fast/intermediate exchange in early points followed by a switch in the behaviour to slow exchange at an intermediate point. This pattern is consistent with a model for multi-step complex assembly via intermediate species formed in fast exchange followed by a co-operative ‘lock-in’ to the final state in slow exchange. The second part of the thesis describes in vitro folding and interaction studies of receptor interacting protein kinase 1-death domain (RIP1-DD) and represents the first biophysical/biochemical characterisation for this protein. The combined results from different techniques suggest that human (but not macaque) RIP1-DD adopts a molten globule state. Interaction studies between RIP1-DD and FADD-DD showed formation of a complex with molecular weight exceeding 190 kDa.

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