MRC National Institute for Medical Research, London
Science for Health
Unpicking the death-inducing signalling complex
12 October 2010
Many cell types in the body undergo regulated cell death, via a process called apoptosis. This can be triggered either by events within the cell itself, such as DNA damage, or by the command of other cells. In the latter case a cell-surface ‘death’ ligand on the ‘executioner’ cell interacts with a transmembrane receptor on the target cell. These receptors are members of the tumour necrosis factor receptor superfamily, also called ‘death receptors’, which contain a globular cytoplasmic domain with an all alpha-helical polypeptide fold: the death domain (DD). Ligand binding to the death receptor leads to the clustering of adaptor proteins and then ‘death effector’ enzymes. The formation of this ‘death-inducing signalling complex’ (DISC) results in the activation of a process that leads to the ordered breakdown of the cell into membrane-enclosed particles (apoptotic bodies) that are engulfed and metabolised by other cells.
Compared to other types of cell-surface receptor (such as those that respond to growth factors or cytokines) the molecular mechanism by which the death receptors operate is unusual in that there does not appear to be a major role for post-translational modification of the receptor. In the case of the canonical death receptor known as CD95 (or Fas), which has an established role in the maintenance of the adaptive immune system, DISC formation is initiated by the interaction of the receptor DD with the adaptor protein FADD (Fas-associated protein with a death domain), which itself contains a DD in its C-terminal half. It has been known for some time that the CD95 and FADD DDs spontaneously form a complex in vitro. In vivo, mutations in the CD95-DD are known to cause a rare autoimmune disease (Type Ia autoimmune lymphoproliferative syndrome, or ALPS) whose patients also exhibit an elevated risk of lymphoma, as a result of reduced CD95-FADD binding.
The structure of the complex between CD95 and FADD DDs has been the subject of intense investigation. In 2009 an X-ray crystal structure was reported for the complex that showed the two DDs in a 4+4 heterooctamer complex that included the CD95 DD with a dramatically altered polypeptide conformation.
Paul Driscoll (pictured) and Katrin Rittinger, in NIMR’s Division of Molecular Structure, have employed state-of-the-art methods in a technique known as heteronuclear nuclear magnetic resonance spectroscopy (NMR) to probe the complex in solution. Together with collaborators in Oxford University, the team has demonstrated that in vitro the complex has a different organisation that comprises ten protein chains in a 5+5 arrangement. Moreover the data unequivocally show that the C-terminus of the CD95 domains is dynamically flexible and not structurally ordered as suggested by the crystal structure. The NMR data are consistent with a complex in which the CD95 chains maintain their canonical overall fold, but appear to occupy slightly different environments suggesting a lack of high order symmetry for the particle.
Speculation that this complex may be similar in structure to an unrelated assembly of DDs from the proteins PIDD and RAIDD is supported by a complementary analysis from the group of Hao Wu at Cornell in New York (Nature Structural and Molecular Biology doi:10.1038/nsmb.1920). Both the NIMR and Cornell teams argue that this complex is better able to explain the available structure-activity data for the interaction including data from disease mutations and various experimental approaches reported to date.
Understanding how CD95 and FADD interact will provide insight into the mechanism of DR signalling and rationalise the molecular basis of ALPS. Moreover, CD95 is homologous to other death receptors that also interact with FADD and show promise as potential anti-cancer targets. Our work serves to clarify the nature of the initial interaction between the DDs of CD95 and FADD and provides a new view of how DISC formation is coupled to death-ligand induced aggregation of death receptors. Collectively these results give rise to the idea that DDs may interact in high order oligomeric assemblies lacking mirror symmetry. It will be interesting to discover if these structures are maintained in the later stages of DISC development when additional effector proteins are recruited.
Paul Driscoll
Click image to view at full-size
Two-dimensional 15N-heteronuclear NMR spectra of CD95-DD (pink) free (left) and mixed (right) with excess unlabelled FADD-DD (green). The result indicates that in the CD95-FADD complex the C-terminal tail region of CD95 remains flexible (inset cartoon).
Original article
Diego Esposito, Andrew Sankar, Nina Morgner, Carol V. Robinson, Katrin Rittinger and Paul C. Driscoll (2010).
Solution NMR investigation of the CD95/FADD homotypic death domain complex suggests lack of engagement of the CD95 C-Terminus
Structure, 18:1378-90. Publisher abstract
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