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We have addressed complex formation between the death domain (DD) of the death receptor CD95 (Fas/APO-1) with the DD of immediate adaptor protein FADD using nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and size-exclusion chromatography with in-line light scattering. We find complexation to be independent of the C-terminal 12 residues of CD95 and insensitive to mutation of residues that engage in the high-order clustering of CD95-DD molecules in a recently reported crystal structure obtained at pH 4. Differential NMR linewidths indicate that the C-terminal region of the CD95 chains remains in a disordered state and (13)C-methyl TROSY data are consistent with a lack of high degree of symmetry for the complex. The overall molecular mass of the complex is inconsistent with that in the crystal structure, and the complex dissociates at pH 4. We discuss these findings using sequence analysis of CD95 orthologs and the effect of FADD mutations on the interaction with CD95.

Original publication

DOI

10.1016/j.str.2010.08.006

Type

Journal article

Journal

Structure

Publication Date

13/10/2010

Volume

18

Pages

1378 - 1390

Keywords

Amino Acid Sequence, Carbon Isotopes, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Fas-Associated Death Domain Protein, Humans, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Mass Spectrometry, Models, Molecular, Molecular Sequence Data, Multiprotein Complexes, Mutation, Nitrogen Isotopes, Protein Binding, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Solutions, fas Receptor