title: Flexible stoichiometry and asymmetry of the PIDDosome core complex by heteronuclear NMR spectroscopy and mass spectrometry.
abstract: Homotypic death domain (DD)-DD interactions are important in the assembly of oligomeric signaling complexes such as the PIDDosome that acts as a platform for activation of caspase-2-dependent apoptotic signaling. The structure of the PIDDosome core complex exhibits an asymmetric three-layered arrangement containing five PIDD-DDs in one layer, five RAIDD-DDs in a second layer and an additional two RAIDD-DDs. We addressed complex formation between PIDD-DD and RAIDD-DD in solution using heteronuclear nuclear magnetic resonance (NMR) spectroscopy, nanoflow electrospray ionization mass spectrometry and size-exclusion chromatography with multi-angle light scattering. The DDs assemble into complexes displaying molecular masses in the range 130-158kDa and RAIDD-DD:PIDD-DD stoichiometries of 5:5, 6:5 and 7:5. These data suggest that the crystal structure is representative of only the heaviest species in solution and that two RAIDD-DDs are loosely attached to the 5:5 core. Two-dimensional (1)H,(15)N-NMR experiments exhibited signal loss upon complexation consistent with the formation of high-molecular-weight species. (13)C-Methyl-transverse relaxation optimized spectroscopy measurements of the PIDDosome core exhibit signs of differential line broadening, cross-peak splitting and chemical shift heterogeneity that reflect the presence of non-equivalent sites at interfaces within an asymmetric complex. Experiments using a mutant RAIDD-DD that forms a monodisperse 5:5 complex with PIDD-DD show that the spectroscopic signature derives from the quasi- but non-exact equivalent environments of each DD. Since this characteristic was previously demonstrated for the complex between the DDs of CD95 and FADD, the NMR data for this system are consistent with the formation of a structure homologous to the PIDDosome core.
authors.affiliation: Division of Molecular Structure, Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Lily A
authors.initials: LA
authors.lastname: Nematollahi
authors.affiliation: Division of Molecular Structure, Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Acely
authors.initials: A
authors.lastname: Garza-Garcia
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.
authors.forename: Chérine
authors.initials: C
authors.lastname: Bechara
authors.affiliation: Division of Molecular Structure, Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Diego
authors.initials: D
authors.lastname: Esposito
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.
authors.forename: Nina
authors.initials: N
authors.lastname: Morgner
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.affiliation: Division of Molecular Structure, Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Electronic address: pdrisco@nimr.mrc.ac.uk.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Driscoll
mesh_terms: D000595:Amino Acid Sequence; D053420:CRADD Signaling Adaptor Protein / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D018360:Crystallography, X-Ray; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism / Q000648:ultrastructure; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D053420:CRADD Signaling Adaptor Protein; C104252:CRADD protein, human; D053418:Death Domain Receptor Signaling Adaptor Proteins; D046912:Multiprotein Complexes; C485896:PIDD1 protein, human
keywords: ILV-labeling; TROSY NMR; death domain; protein complex; spectral complexity