The death inducing signaling complex (DISC) formed by the death receptor Fas, the adapter protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas–FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5–7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas–FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrence of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects of Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher order aggregation.

Biophysical studies of membrane proteins are often impeded by the requirement for a membrane mimicking environment. Detergent micelles are the most common choice, but the denaturing properties make them unsatisfactory for studies of many membrane proteins and their interactions. In the present work, we explore phospholipid bilayer nanodiscs as membrane mimics and employ electron microscopy and solution NMR spectroscopy to characterize the structure and function of the human voltage dependent anion channel (VDAC-1) as an example of a polytopic integral membrane protein. Electron microscopy reveals the formation of VDAC-1 multimers, an observation that is consistent with results obtained in native mitochondrial outer membranes. High-resolution NMR spectroscopy demonstrates a well folded VDAC-1 protein and native NADH binding functionality. The observed chemical shift changes upon addition of the native ligand NADH to nanodisc-embedded VDAC-1 resemble those of micelle-embedded VDAC-1, indicating a similar structure and function in the two membrane-mimicking environments. Overall, the ability to study integral membrane proteins at atomic resolution with solution NMR in phospholipid bilayers, rather than in detergent micelles, offers exciting novel possibilities to approach the biophysical properties of membrane proteins under non-denaturing conditions, which makes this technology particular suitable for protein-protein interactions and other functional studies.Membrane proteins are responsible for a wide range of essential physiological processes and are involved in various diseases. Most biophysical methods for studying membrane proteins in vitro require a membrane mimicking environment to stabilize the protein. 1 The most commonly used membrane mimics are detergents, but since they often have deteriorating effects on the structure and activity of a protein, the search for a suitable detergent is an empirical and thus time-consuming process. A better mimic are phospholipid bilayers, since their biophysical properties resemble more closely those of the native membrane and they are thus more likely to maintain membrane proteins in a stable and active state. Phospholipid bilayers in the form of bicelles have been used to study membrane proteins by high resolution biophysical methods such as NMR spectroscopy 2 and x-ray crystallography. 3 However, the morphology of bicelles is complex, depending on various sample conditions, such as gerhard_wagner@hms.harvard.edu. Supporting Information Available: Experimental procedures and data on the purification, refolding and reconstitution of VDAC-1 into nanodiscs. This material is available free of charge via the Internet at http://pubs.acs.org. A promising new approach for studies of membrane proteins in a phospholipid bilayer uses high-density lipoprotein nanodiscs consisting of a central disk-shaped core of lipids, confined by two copies of an α-helical amphipathic protein. [5][6][7] These nanoscale lipid bilayers with reported diameters ranging from 9.5 to 12 nm prov...

SUMMARY Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Non-conventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here we present a comparative study of the hepta-helical membrane protein bacteriorhodopsin in detergent micelles, amphipols and nanodiscs. Our results confirm that non-conventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are in principle accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguishes regions of bacteri-orhodopsin that mediate membrane/solvent contacts in the tested environments whereas the protein’s functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.