Abstract
Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E. coli and HeLa cell extracts. We found that the gem-diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.
Highlights
Apoptosis is a form of programmed cell death pivotal for mammals
We addressed the compatibility of the different labels with Bax fold and activity, and the stability of the spin-labeled Bax variants in a cellular context using E. coli or HeLa cell extracts
In this study we used Bax wild type (BaxWT), which has two native cysteines at positions 62 and 126 and the mutant BaxC87(C62S, C126S) which has a single surface-exposed cysteine (Fig. 1d). We chose these two MTSL-labeled variants because we previously showed that they are active and well folded in both inactive and active conformations and their interspin distance distributions are clearly distinguishable in both conformations[18,23]
Summary
Apoptosis is a form of programmed cell death pivotal for mammals. Aberrant apoptosis is involved in severe diseases like cancer, stroke, myocardial infarction or autoimmune, as well as neurodegenerative diseases[1,2,3]. The interaction of the pro- and anti-apoptotic Bcl-2 protein partners plays a crucial role in apoptosis regulation and execution[4,5,6]. We focus on the pro-apoptotic Bcl-2 protein Bax, which exists in two major conformations In healthy cells it is mainly a soluble, monomeric, inactive protein whose structure is known[12], while upon a pro-apoptotic stimulus Bax transforms into the active pore-forming, membrane-embedded oligomer, whose structure is only partially known (recently reviewed in[13]). To simplify the spin system and obtain distance constraints within each monomer in the oligomer, we used doubly-labeled proteins spin-diluted with unlabeled wild type partners[18], which enhanced the intra-monomer distance within the distance distribution Based on these data we could propose a coarse-grained model of active Bax[13]. The conventional nitroxide labels were found to be incompatible with cellular extracts or isolated mitochondria, as intra-cellular agents can chemically reduce the nitroxide group to the EPR silent hydroxylamine or can release the labels from the protein after reduction of the S-S linker[18,30]
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