Solution NMR Investigation of the CD95/FADD Homotypic Death Domain Complex Suggests Lack of Engagement of the CD95 C Terminus

• Published in: Structure (London) 2010-10, Vol.18 (10), p.1378-1390
• Main Authors: Esposito, Diego, Sankar, Andrew, Morgner, Nina, Robinson, Carol V., Rittinger, Katrin, Driscoll, Paul C.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Carbon Isotopes; Complex formation; Complexation; Crystal structure; Crystallography, X-Ray; Death; Electrophoresis, Polyacrylamide Gel; fas Receptor - chemistry; fas Receptor - genetics; fas Receptor - metabolism; Fas-Associated Death Domain Protein - chemistry; Fas-Associated Death Domain Protein - genetics; Fas-Associated Death Domain Protein - metabolism; Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy - methods; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Multiprotein Complexes - chemistry; Multiprotein Complexes - metabolism; Mutation; Mutations; Nitrogen Isotopes; Nuclear magnetic resonance; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Residues; Sequence Homology, Amino Acid; Solutions
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2010.08.006

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 13C-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. [Display omitted] ► NMR-focused investigation of CD95/Fas suggests that their two death domains (DDs) form a complex in solution ► DD complex comprises either nine or ten domains in 5:4 or 5:5 ratio ► DD complex retains flexibility of the C-terminal tail of CD95/Fas and lacks a high degree of symmetry ► The data suggest that the conformation of the CD95/Fas DD is remodeled in the complex