Structural Interactions Of Death Domains In The RIP1 And FADD Complex For Cell Necrosis

Cell death is a natural phenomenon in maintaining the normal morphology,function and growth of living organisms.Cell necrosis is one of the pathways of cell death.Different from apoptosis,cell necrosis can cause cell damage but it was still tightly controlled by a battery of molecular regulators in vivo in a process known as programmed cell death.Cell necrosis can be mediated by death receptors such as FasL and TNFR.This thesis focuses on the structural studies of the complex formed by the death domains of RIP1 and FADD in the signaling pathway mediated by the death receptor TNFR1(tumor necrosis factor receptor 1)for programmed cell death.In previous studies,it was shown that recombinant human RIP 1-Death domain(RIP1-DD)interacts with FADD-DD in vitro.In this thesis,a series of recombinant plasmids encoding human RIP1-DD and FADD-DD were used for the prokaryotic expression.Attempts were made to form the complex by co-expression or mixing the newly expressed proteins.The protein complex purified by chromatography was with a molecular weight of 276.9kDa based on multi-angle light scattering experiments.Dynamic light scattering experiments show that the protein solution is a moderately dispersed system with good homogeneity.Multiple types,including a rod-shaped and a rhombic polyhedron-shaped,crystals were obtained through initial screening and optimization.From a diffracting crystal,we determined the crystal structure of hRIP1DD(577-669)monomer by molecular replacement(MR)in combination with singlewavelength anomalous scattering(SAD)to 2.6 ? resolution.Based on the analysis of this hRIPl-DD structure,an initial interacting model was built.The residues on the putative interacting interface were mutated for GST pulldown experiments to confirm their roles in maintaining the inter-protein interaction.This work laid a foundation for the further structural studies of the RIP1-DD and FADD-DD complex.