Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association.

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.

A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels.

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.

gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility.

The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins' fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the "true" biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.

gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility.

Invited for this month's cover picture is the group of Professor Enrica Bordignon at the Ruhr University Bochum. The cover picture shows an artistic view of E. coli cells and two spin-labeled recombinantly produced proteins, which can be inserted into the cells for EPR studies. The primary sequence of the proteins is schematically shown with the one-letter amino acid code, and cysteine residues are functionalized with the two new gem diethyl nitroxide spin labels designed to better sustain the reducing cellular environment. Read the full text of their Full Paper at 10.1002/open.201900119.

Topology of active, membrane-embedded Bax in the context of a toroidal pore.

Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into the outer mitochondrial membrane, oligomerizes, and forms pores that release proapoptotic factors like Cytochrome c into the cytosol. The structures of active Bax and its homolog Bak are only partially understood and the topology of the proteins with respect to the membrane bilayer is controversially described in the literature. Here, we systematically review and examine the protein-membrane, protein-water, and protein-protein contacts of the nine helices of active Bax and Bak, and add a new set of topology data obtained by fluorescence and EPR methods. We conclude based on the consistent part of the datasets that the core/dimerization domain of Bax (Bak) is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane. Among the available structural models, those considering the dimerization/core domain at the rim of a toroidal pore are the most plausible to describe the active state of the proteins, although the structural flexibility of the piercing/latch domain does not allow unambiguous discrimination between the existing models.

Bcl-2 proteins: Unraveling the details of a complex and dynamic network.

Apoptosis regulation by Bcl-2 proteins is pivotal for mammalians, not only because it is key for development but also because aberrant apoptosis is prerequisite to severe diseases, like cancer. Recently, we quantified interactions within the Bcl-2 protein network in solution and membranes, and addressed membrane recruitment, preference of interaction partners and the consequences for Bax activation and inhibition.

Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes for apoptosis regulation.

The Bcl-2 proteins form a complex interaction network that controls mitochondrial permeabilization and apoptosis. The relative importance of different Bcl-2 complexes and their spatio-temporal regulation is debated. Using fluorescence cross-correlation spectroscopy to quantify the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, we show that membrane insertion drastically alters the pattern of Bcl-2 complexes, and that the C-terminal helix of Bcl-xL determines its binding preferences. At physiological temperature, Bax can spontaneously activate in a self-amplifying process. Strikingly, Bax also recruits Bcl-xL to membranes, which is sufficient to retrotranslocate Bax back into solution to secure membrane integrity. Our study disentangles the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation.The permeabilization of the mitochondrial outer membrane to induce apoptosis is regulated by complex interactions between Bcl-2 family members. Here the authors develop a quantitative interactome of a membrane Bcl-2 network and identify a hierarchy of protein complexes in apoptosis induction.

The membrane activity of BOK involves formation of large, stable toroidal pores and is promoted by cBID.

The BCL-2 family members are key regulators of the intrinsic apoptotic pathway, which is defined by permeabilization of the mitochondrial outer membrane by members of the BAX-like subfamily. BOK is classified as a BAX-like protein; however, its (patho-)physiological role remains largely unclear. We therefore assessed the membrane permeabilization potential of C-terminally truncated recombinant BOK, BOK∆C . We show that BOK∆C can permeabilize liposomes mimicking the composition of mitochondrial outer membrane, but not of endoplasmic reticulum, forming large and stable pores over time. Importantly, pore formation was enhanced by the presence of cBID and refractory to the addition of antiapoptotic BCL-XL . However, isolated mitochondria from Bax-/- Bak-/- cells were resistant to BOK-induced cytochrome c release, even in the presence of cBID. Taken together, we show that BOK∆C can permeabilize liposomes, and cooperate with cBID, but its role in directly mediating mitochondrial permeabilization is unclear and may underlie a yet to be determined negative regulation.

Pushing the size limit of de novo structure ensemble prediction guided by sparse SDSL-EPR restraints to 200 residues: The monomeric and homodimeric forms of BAX.

Structure determination remains a challenge for many biologically important proteins. In particular, proteins that adopt multiple conformations often evade crystallization in all biologically relevant states. Although computational de novo protein folding approaches often sample biologically relevant conformations, the selection of the most accurate model for different functional states remains a formidable challenge, in particular, for proteins with more than about 150 residues. Electron paramagnetic resonance (EPR) spectroscopy can obtain limited structural information for proteins in well-defined biological states and thereby assist in selecting biologically relevant conformations. The present study demonstrates that de novo folding methods are able to accurately sample the folds of 192-residue long soluble monomeric Bcl-2-associated X protein (BAX). The tertiary structures of the monomeric and homodimeric forms of BAX were predicted using the primary structure as well as 25 and 11 EPR distance restraints, respectively. The predicted models were subsequently compared to respective NMR/X-ray structures of BAX. EPR restraints improve the protein-size normalized root-mean-square-deviation (RMSD100) of the most accurate models with respect to the NMR/crystal structure from 5.9Å to 3.9Å and from 5.7Å to 3.3Å, respectively. Additionally, the model discrimination is improved, which is demonstrated by an improvement of the enrichment from 5% to 15% and from 13% to 21%, respectively.

Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species.

Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in <1 min. Strikingly, active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

Formation of disulfide bridges drives oligomerization, membrane pore formation, and translocation of fibroblast growth factor 2 to cell surfaces.

Fibroblast growth factor 2 (FGF2) is a key signaling molecule in tumor-induced angiogenesis. FGF2 is secreted by an unconventional secretory mechanism that involves phosphatidylinositol 4,5-bisphosphate-dependent insertion of FGF2 oligomers into the plasma membrane. This process is regulated by Tec kinase-mediated tyrosine phosphorylation of FGF2. Molecular interactions driving FGF2 monomers into membrane-inserted FGF2 oligomers are unknown. Here we identify two surface cysteines that are critical for efficient unconventional secretion of FGF2. They represent unique features of FGF2 as they are absent from all signal-peptide-containing members of the FGF protein family. We show that phosphatidylinositol 4,5-bisphosphate-dependent FGF2 oligomerization concomitant with the generation of membrane pores depends on FGF2 surface cysteines as either chemical alkylation or substitution with alanines impairs these processes. We further demonstrate that the FGF2 variant forms lacking the two surface cysteines are not secreted from cells. These findings were corroborated by experiments redirecting a signal-peptide-containing FGF family member from the endoplasmic reticulum/Golgi-dependent secretory pathway into the unconventional secretory pathway of FGF2. Cis elements known to be required for unconventional secretion of FGF2, including the two surface cysteines, were transplanted into a variant form of FGF4 without signal peptide. The resulting FGF4/2 hybrid protein was secreted by unconventional means. We propose that the formation of disulfide bridges drives membrane insertion of FGF2 oligomers as intermediates in unconventional secretion of FGF2.

Structural model of active Bax at the membrane.

Bax plays a central role in the mitochondrial pathway of apoptosis. Upon activation, cytosolic Bax monomers oligomerize on the surface of mitochondria and change conformation concertedly to punch holes into the outer membrane. The subsequent release of cytochrome c initiates cell death. However, the structure of membrane-inserted Bax and its mechanism of action remain largely unknown. Here, we propose a 3D model of active Bax at the membrane based on double electron-electron resonance (DEER) spectroscopy in liposomes and isolated mitochondria. We show that active Bax is organized at the membrane as assemblies of dimers. In addition to a stable dimerization domain, each monomer contains a more flexible piercing domain involved in interdimer interactions and pore formation. The most important structural change during Bax activation is the opening of the hairpin formed by helices 5 and 6, which adopts a clamp-like conformation central to the mechanism of mitochondrial permeabilization.

New biophysical methods to study the membrane activity of Bcl-2 proteins.

The proteins of Bcl-2 family are key regulators of apoptosis. Many Bcl-2 proteins have the unique ability to switch between two possible conformations, soluble in the cytosol or associated to cellular membranes. Importantly, their membrane-inserted form is the main responsible for their apoptotic function. Unfortunately, there are only a limited number of methods available to study the membrane activity of these proteins. Here, we present a methodology to study protein binding to membranes and membrane permeabilization at the single vesicle level. It is based on purified proteins and giant unilamellar vesicles and involves directly visualization of the process with a confocal microscope. This approach allows for the characterization of the membrane activity of the Bcl-2 proteins (or of any other pore-forming molecule) with unprecedented detail.

Automated analysis of giant unilamellar vesicles using circular Hough transformation.

MOTIVATION: In order to obtain statistically relevant results, the study of membrane effects at the single-vesicle level requires the analysis of several hundreds of giant unilamellar vesicles (GUVs), which becomes a very time-consuming task if carried out manually. Complete and user-friendly software for fast and bias-free automated analysis has not been reported yet. RESULTS: We developed a framework for the automated detection, tracking and analysis of individual GUVs on digital microscopy images. Our tool is suited to quantify protein binding to membranes as well as several aspects of membrane permeabilization on single vesicles. We demonstrate the applicability of the approach by comparing alternative activation methods for Bax, a pore-forming protein involved in mitochondrial permeabilization during apoptosis. AVAILABILITY AND IMPLEMENTATION: The complete software is implemented in MATLAB (The MathWorks, Inc., USA) and available as a standalone as well as the full source code at http://www.ifib.uni-tuebingen.de/research/garcia-saez/guv-software.html.

Proapoptotic Bax and Bak proteins form stable protein-permeable pores of tunable size.

The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e. Smac/DIABLO, AIF, and endoglycosidase G). However, the properties and nature of Bax/Bak apoptotic pores remain enigmatic. Here, we performed a detailed analysis of the membrane permeabilizing activity of Bax and Bak at the single vesicle level. We directly visualized that cBid-activated Bax and BakΔC21 can form membrane pores large enough to release not only cytochrome c, but also allophycocyanine, a protein of 104 kDa. Interestingly, the size of Bax and BakΔC21 pores is not constant, as typically observed in purely proteinaceous channels, but evolves with time and depends on protein concentration. We found that Bax and BakΔC21 formed long-lived pores, whose areas changed with the amount of Bax/BakΔC21 but not with cardiolipin concentration. Altogether, our results demonstrate that Bax and BakΔC21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores.

Mechanistic differences in the membrane activity of Bax and Bcl-xL correlate with their opposing roles in apoptosis.

Based on their membrane-permeabilizing activity in vitro, it has been proposed that Bax-like proteins induce cytochrome c release during apoptosis via pore formation. However, antiapoptotic Bcl-2 proteins, which inhibit cytochrome c release, also display pore activity in model membranes. As a consequence, a unified description that aligns the pore activity of the Bcl-2 proteins with their apoptotic function is missing. Here, we studied the mechanism of membrane binding, oligomerization, and permeabilization by pro- and antiapoptotic Bcl-2 members at the single-vesicle level. We found that proapoptotic Bax forms large, stable pores via an all-or-none mechanism that can release cytochrome c. In contrast, antiapoptotic Bcl-xL induces transient permeability alterations in pure lipid membranes that have no consequences for the mitochondrial outer membrane but inhibit Bax membrane insertion. These differences in pore activity correlate with a distinct oligomeric state of Bax and Bcl-xL in membranes and can be reproduced in isolated mitochondria. Based on our findings, we propose new models for the mechanisms of action of Bax and Bcl-xL that relate their membrane activity to their opposing roles in apoptosis and beyond.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion.

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.

Dynamic interaction of cBid with detergents, liposomes and mitochondria.

The BH3-only protein Bid plays a key role in the induction of mitochondrial apoptosis, but its mechanism of action is still not completely understood. Here we studied the two main activation events of Bid: Caspase-8 cleavage and interaction with the membrane bilayer. We found a striking reversible behaviour of the dissociation-association events between the Bid fragments p15 and p7. Caspase-8 cleavage does not induce per se separation of the two Bid fragments, which remain in a stable complex resembling the full length Bid. Detergents trigger a complete dissociation, which can be fully reversed by detergent removal in a range of protein concentrations from 100 µM down to 500 nM. Incubation of cBid with cardiolipin-containing liposomes leads to partial dissociation of the complex. Only p15 (tBid) fragments are found at the membrane, while p7 shows no tendency to interact with the bilayer, but complete removal of p7 strongly increases the propensity of tBid to become membrane-associated. Despite the striking structural similarities of inactive Bid and Bax, Bid does not form oligomers and reacts differently in the presence of detergents and membranes, highlighting clear differences in the modes of action of the two proteins. The partial dissociation of cBid triggered by the membrane is suggested to depend on the strong and specific interaction between p15 and p7. The reversible disassembly and re-assembly of the cBid molecules at the membrane was as well proven by EPR using spin labeled cBid in the presence of isolated mitochondria. The observed dynamic dissociation of the two Bid fragments could allow the assistance to the pore-forming Bax to occur repeatedly and may explain the proposed "hit-and-run" mode of action of Bid at the bilayer.

Quantification of protein-protein interactions within membranes by fluorescence correlation spectroscopy.

The characterization of interactions between membrane proteins as they take place within the lipid bilayer poses a technical challenge, which is currently very difficult and, in many cases, impossible to overcome. The recent development of a method based in the combination two-color fluorescence cross-correlation spectroscopy with scanning of the focal volume allows the detection and quantification of interactions between biomolecules inserted in biological membranes. This powerful strategy has allowed the quantitative analysis of diverse systems, such as the association between proteins of the Bcl-2 family involved in apoptosis regulation or the binding between a growth factor and its receptor during signaling. Here, we review the last developments to quantify protein/protein interactions in lipid membranes and focus on the use of fluorescence-correlation-spectroscopy approaches for that purpose.