Solution NMR investigation of the CD95/FADD homotypic death domain complex suggests lack of engagement of the CD95 C terminus.
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.
Division of Molecular Structure, MRC-National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Diego
D
Esposito
Andrew
A
Sankar
Nina
N
Morgner
Carol V
CV
Robinson
Katrin
K
Rittinger
Paul C
PC
Driscoll
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D053200:Fas-Associated Death Domain Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009587:Nitrogen Isotopes; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D019014:fas Receptor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002247:Carbon Isotopes; D053200:Fas-Associated Death Domain Protein; D046912:Multiprotein Complexes; D009587:Nitrogen Isotopes; D012996:Solutions; D019014:fas Receptor