Structural Basis of Fas-Mediated Programmed Cell Death

Rockefeller University, New York NY

Investigators

Abstract

Programmed cell death (PCD) is a normal part of the life and death of a cell. PCD can be triggered by a wide variety of stimuli, such as growth factor deprivation, exposure to mutagenic agents, ionizing radiation, etc. whose stimulus is mediated by a member of the tumor necrosis factor receptor (TNFR) superfamily. A subset of TNFRs, termed death receptors (DRs), are responsible for transducing a death signal into a cellular response. DRs are characterized by the presence of a C-terminal six-helix bundle known as the death domain (DD). The DD is the focal point of the assembly of an oligomeric complex around which assemble a group of proteins that sense the death signal and transmit the signal to send it down a signaling cascade. One of the most important DRs is the Fas/CD95 receptor. Fas plays a role in the regulation of the immune response and in the control of peripheral B and T cell survival that is critical to immune cell homeostasis. Fas-mediated PCD is implicated in the maintenance of immune privilege in the eye and testis as well as in attenuating immunosurveillance of certain types of tumors. In response to a stimulus, Fas is thought to oligomerize through the DD and recruit the effector FADD. Like Fas, FADD contains a DD at its C-terminus and binds, albeit with unknown stoichiometry, to the receptor DD. On the N-terminus of FADD is a second protein-interaction domain, the death effector domain (DED), which recruits the initiator caspase-8 to the growing signalling complex. The Fas/FADD/caspase-8 complex is known as the death-inducing signalling complex (DISC) and represents the minimal complex necessary for induction of PCD. Although the structure of the FADD-DD and FADD-DED have been solved in truncated forms by NMR, there is yet to be a clear description of the binding surface, binding affinity or binding stoichiometry of FADD to Fas in an activated state. Indeed, this information is lacking for all DRs identified to date. This project will conduct a series of biophysical studies aimed at defining the molecular architecture of FADD in the Fas signaling complex. Specifically, the relative orientation of the FADD DD and DED will be determined from completion of the full-length 3D structure of FADD in solution. The stoichiometry of the Fas/FADD interaction will be measured by gel-filtration, analytical ultracentrifugation and isothermal binding calorimetry of the complex using a soluble form of the Fas receptor. It will further be determined whether the DD is sufficient for DISC assembly by construction of a soluble receptor mimic with a defined oligomerization state for the Fas-DD. Finally, mutagenesis in combination with isothermal binding calorimetry and analytical ultracentrifugation will define the binding surface of the Fas/FADD complex and determine the binding affinity. Collectively these data will provide new insights into the architecture of the Fas DISC. The outcome of this study will provide a molecular insight into the mechanism of programmed cell death.