The Structural Basis Of DR3 Intracellular Signal Activation

The TNFRSF(tumor necrosis factor receptor super family)is one of the most important protein families in immune system and their members have a wide spectrum of immunological functions including organization of lymphoid tissue development,co-stimulation of lymphocytes,and lymphocyte cell fate determination.Belonging to the same protein family,the DR3 shows high structural similarity to TNFR1.Both of them have four repeated cysteine-rich domains(CRDs)in extracellular region and a death domain in their intracellular domain.The predominant response of DR3 upon its ligand TNF-like protein 1A(TL1A)binding is a pro-inflammatory and survival-promoting signal cascade.Mechanistically,TL1A-DR3 interaction initiates signal transduction through the adaptor protein TNFR-associated death domain(TRADD),which promotes activation of NF-κB signaling.Death domain belongs to DD superfamily(DDS)and it is featured by conserved homotypic interaction motifs which are constituted by six amphipathic a-helices folded in an antiparallel a-helical bundle.DR3 DD consists of around 80 amino acids and can transmit signals through homotypic dimerization and apical protein complexes nucleated by the DD of adaptor TRADD.TRADD consists of two domains,an N-terminal domain and a DD domain in C terminus.The protein complex of N-terminal domain from TRADD and TRAF domain from TRAF2(TNF receptor-associated factor 2)has been determined,which elucidated the subsequent signal complex of both DR3 and TNFR1 signal pathways.However,how does extracellular ligand engagement of DR3 cross cell membrane and deliver the activation signal to its intracellular domain,recruit its adaptor TRADD and further initiate downstream signaling cascades still remains to be elucidated.There are three main results in this thesis:(1)We determined crystal structures of both moues and human DR3 DD and they displayed completely different fold.Mouse DR3 DD adopts a six-helix bundle structure as classical death domain whereas human DR3 DD displayed non-classical structure.It has only 5 helices and lacks of a hydrophobic core with a long helix in the middle,composed of H3 and H4 corresponding to moues DR3 DD structure.An extensive hydrophobic homotypic interaction interface was found in the human DR3 DD crystal.Through yeast two-hybrid,mammalian two-hybrid,NF-κB luciferase assay and pull-down assay,we showed both human and mouse DR3 DD share similar solution conformations and functions in cells.Hence,we hypothesize that the two different structures represent two conformations of DR3 DD,namely open conformation and closed conformation.(2)DR3 DD can undergo self-interaction,which is essential for signaling.Through yeast two-hybrid,mammalian two-hybrid,NF-κB luciferase assay,pull-down assay and small angle X-ray scattering,we identified critical sites of DR3 DD for its homotypic interaction and interaction with TRADD and found its open conformation is required for signal transduction.(3)Through yeast two-hybrid,mammalian two-hybrid and pull-down assay DR3,we identified DD tends to bind with TRADD using open conformation through core binding site on TRADD which is located on β-hairpin motif according to the NMR structure of C-terminal region of TRADD.Based on the results of this thesis,a model is proposed:the DD region is folded as a classical six-helix bundle in resting state and the open conformation of DR3 DD is more accessible for its self-interaction upon activation,which facilitates the recruitment of TRADD.Furthermore,a highly conserved β hairpin motif,which is immediately adjacent to the helical region in C-terminal domain of TRADD,harbors critical binding sites for DR3 DD.This study presents a novel structural switch model of DR3 activation,which allows NF-κB activation and downstream inflammatory regulation.