Neutron reflectometry and NMR spectroscopy of full-length Bcl-2 protein reveal its membrane localization and conformation

Abstract

B-cell lymphoma 2 (Bcl-2) proteins are the main regulators of mitochondrial apoptosis. Anti-apoptotic Bcl-2 proteins possess a hydrophobic tail-anchor enabling them to translocate to their target membrane and to shift into an active conformation where they inhibit pro-apoptotic Bcl-2 proteins to ensure cell survival. To address the unknown molecular basis of their cell-protecting functionality, we used intact human Bcl-2 protein natively residing at the mitochondrial outer membrane and applied neutron reflectometry and NMR spectroscopy. Here we show that the active full-length protein is entirely buried into its target membrane except for the regulatory flexible loop domain (FLD), which stretches into the aqueous exterior. The membrane location of Bcl-2 and its conformational state seems to be important for its cell-protecting activity, often infamously upregulated in cancers. Most likely, this situation enables the Bcl-2 protein to sequester pro-apoptotic Bcl-2 proteins at the membrane level while sensing cytosolic regulative signals via its FLD region.

Unravelling the structural complexity of protein–lipid interactions with neutron reflectometry

“…The examination of lipid nanodiscs with and without embedded proteins at interfaces has been an area of interest for reflectometry studies in recent years [42][43][44] [45] , with peptide coated nanodiscs being used recently to create supported lipid bilayers containing well orientated integral membrane proteins [46] . NR examination of model membrane sample systems can complement studies using atomic force microscopy [47] , solid state NMR spectroscopy [48] , quartz crystal microbalance measurements [49] , Cryo-electron microscopy [50] , surface plasmon resonance [51] and ellipsometry [52] with NR, uniquely, providing the complex structure across the membrane. Neutrons are reflected and refracted at interfacial scattering length density boundaries such as the solid/liquid, air/liquid or solid/air interfaces (Fig 1, B).…”

“…Currently this has been used to benchmark biotechnological sensor systems [67] or new integral membrane protein containing membrane sample systems [68][46] . Biochemical structural studies on integral membrane proteins provide a means of structurally probing function within the membrane environment [48][69][70] [71] .…”

Bax Forms a Membrane Surface Protein-Lipid Complex as it Initiates Apoptosis

“…Since those proteins have to be membrane-active to interact at the MOM and control its integrity, they possess the typical features of "tail anchored membrane proteins" with an amphitropic main globular fold and a single transmembrane (TM) domain to enable association to and penetration into the MOM target membrane (5,6). While Bcl-2 is insoluble with its TM domain stretched out to anchor and immerse the protein into its membrane environment (7), Bax is soluble by hiding its TM domain inside its hydrophobic groove region (8).…”

“…During this process the mitochondrial shell – its mitochondrial outer membrane (MOM) - undergoes permeabilization, thereby enabling release of apoptotic factors such as cytochrome which triggers an irreversible signaling cascade (6). To protect healthy cells from undesired clearance, this pathway is tightly regulated by the Bcl-2 (B-cell lymphoma 2) protein family (5, 7-9). Pro- and anti-apoptotic members of this family meet at the MOM and they arbitrate the cell’s fate there: intact membrane and survival versus permeabilization and death (1, 2).…”

Mutually Beneficial Combination of Molecular Dynamics Computer Simulations and Scattering Experiments

“…By simply mixing H O and D O, one can adjust the contrast with precision [ 45 , 46 ]. More complex isotopic substitution schemes, for instance at specific molecular sites, can also be used to achieve more targeted control [ 47 , 48 ].…”