Apoptosis, a form of programmed cell death, is a key mechanism in multicellular organisms. Not only does it shape embryos and balances cell proliferation, but it is also an important tool for the immune system removing potentially harmful cells. As important as apoptosis is for an organism, as harmful it is when it does not work correctly. Cells which cannot perform apoptosis can grow unhindered leading to tumors while cells that perform apoptosis without it being necessary contribute to degenerative diseases such as Alzheimer’s disease.

An important part of balancing the mitochondrial pathway of apoptosis in cells is the Bcl-2 protein family. It consists of pro-apoptotic proteins such as Bax and cBid and of anti-apoptotic proteins, such as Bcl-xL which inhibits the activation of Bax by direct inhibition of Bax and/or indirect inhibition of the activator cBid. However, the heterocomplexes between pro- and anti-apoptotic partners are not yet structurally characterized.

To allow biophysical analysis, we purified and characterized full-length monomeric Bcl-xL showing its activity in direct and indirect inhibition of pore formation. We used continuous wave EPR to monitor changes in side-chain dynamics of spin-labeled cBid and Bcl-xL during formation of the inhibitory complex and double electron-electron resonance (DEER) to obtain distance constraints in the complex. The topology of the proteins with respect to the membrane was investigated by electron spin echo envelope modulation (ESEEM). By combining the EPR data with molecular modeling, we could unveil the dynamic structure of the inhibitory Bcl-xL/cBid complex at the membrane, which paves the way for the full characterization of the Bcl-2 interactome.