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High-resolution analysis of the conformational transition of pro-apoptotic Bak at the lipid membrane.

ABSTRACT: Permeabilization of the outer mitochondrial membrane by pore-forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro-apoptotic Bak during pore formation, high-resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX-MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high-resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3-only proteins. Furthermore, we determined the first high-resolution structure of the Bak transmembrane helix. Upon activation, α-helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane-bound state. In line with this finding, comparative protein folding experiments with Bak and anti-apoptotic BclxL suggest that α-helix 1 in Bak is a metastable structural element contributing to its pro-apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α-helix 1 yielded Bak variants with delayed pore-forming activity. These insights will contribute to a better mechanistic understanding of Bak-mediated membrane permeabilization.

SUBMITTER: Sperl LE

PROVIDER: S-EPMC8521305 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Dynamic re-configuration of pro-apoptotic BAK on membranes

Project description:BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.

| S-SCDT-EMBOJ-2020-107237 | biostudies-other

Dynamic re-configuration of pro-apoptotic BAK on membranes

Project description:BAK and BAX, the effectors of intrinsic apoptosis, undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms that describe this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) on liposomes comprising mitochondrial lipids. The HDX-MS profile of BAK on a membrane was broadly consistent with the known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis led by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains BAK in its inactive conformation and disrupting this was sufficient for constitutive BAK activation. Moreover, the entire BAK N-terminus that precedes the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Cleavage of the N-terminus potentiated BAK-mediated membrane permeabilisation on liposomes and mitochondria. Together, HDX-MS reveals new insights into the dynamic nature of BAK activating conformation change in a membrane that will reveal new opportunities for therapeutic targeting.

2021-08-09 | PXD027288 | Pride

Mechanistic picture for conformational transition of a membrane transporter at atomic resolution.

Project description:During their transport cycle, ATP-binding cassette (ABC) transporters undergo large-scale conformational changes between inward- and outward-facing states. Using an approach based on designing system-specific reaction coordinates and using nonequilibrium work relations, we have performed extensive all-atom molecular dynamics simulations in the presence of explicit membrane/solvent to sample a large number of mechanistically distinct pathways for the conformational transition of MsbA, a bacterial ABC exporter whose structure has been solved in multiple functional states. The computational approach developed here is based on (i) extensive exploration of system-specific biasing protocols (e.g., using collective variables designed based on available low-resolution crystal structures) and (ii) using nonequilibrium work relations for comparing the relevance of the transition pathways. The most relevant transition pathway identified using this approach involves several distinct stages reflecting the complex nature of the structural changes associated with the function of the protein. The opening of the cytoplasmic gate during the outward- to inward-facing transition of apo MsbA is found to be disfavored when the periplasmic gate is open and facilitated by a twisting motion of the nucleotide-binding domains that involves a dramatic change in their relative orientation. These results highlight the cooperativity between the transmembrane and the nucleotide-binding domains in the conformational transition of ABC exporters. The approach introduced here provides a framework to study large-scale conformational changes of other membrane transporters whose computational investigation at an atomic resolution may not be currently feasible using conventional methods.

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High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane.

Project description:Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo-electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the ? subunit of the rotor. Assembly of the enzyme with the F6-? fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.

| S-EPMC5948177 | biostudies-literature

Charge profile analysis reveals that activation of pro-apoptotic regulators Bax and Bak relies on charge transfer mediated allosteric regulation.

Project description:The pro-apoptotic proteins Bax and Bak are essential for executing programmed cell death (apoptosis), yet the mechanism of their activation is not properly understood at the structural level. For the first time in cell death research, we calculated intra-protein charge transfer in order to study the structural alterations and their functional consequences during Bax activation. Using an electronegativity equalization model, we investigated the changes in the Bax charge profile upon activation by a functional peptide of its natural activator protein, Bim. We found that charge reorganizations upon activator binding mediate the exposure of the functional sites of Bax, rendering Bax active. The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction. We further identified a network of charge reorganizations that confirms previous speculations of allosteric sensing, whereby the activation information is conveyed from the activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. The network was mediated by a hub of three residues on helix 5 of the hydrophobic core of Bax. Sequence and structural alignment revealed that this hub was conserved in the Bak amino acid sequence, and in the 3D structure of folded Bak. Our results suggest that allostery mediated by charge transfer is responsible for the activation of both Bax and Bak, and that this might be a prototypical mechanism for a fast activation of proteins during signal transduction. Our method can be applied to any protein or protein complex in order to map the progress of allosteric changes through the proteins' structure.

| S-EPMC3375244 | biostudies-literature

MCMV-mediated inhibition of the pro-apoptotic Bak protein is required for optimal in vivo replication.

Project description:Successful replication and transmission of large DNA viruses such as the cytomegaloviruses (CMV) family of viruses depends on the ability to interfere with multiple aspects of the host immune response. Apoptosis functions as a host innate defence mechanism against viral infection, and the capacity to interfere with this process is essential for the replication of many viruses. The Bcl-2 family of proteins are the principle regulators of apoptosis, with two pro-apoptotic members, Bax and Bak, essential for apoptosis to proceed. The m38.5 protein encoded by murine CMV (MCMV) has been identified as Bax-specific inhibitor of apoptosis. Recently, m41.1, a protein product encoded by the m41 open reading frame (ORF) of MCMV, has been shown to inhibit Bak activity in vitro. Here we show that m41.1 is critical for optimal MCMV replication in vivo. Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak(-/-) mice. Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection. The results show that Bax and Bak mediate non-redundant functions during MCMV infection and that the virus produces distinct inhibitors for each protein to counter the activity of these proteins.

| S-EPMC3585157 | biostudies-literature

Conformational changes and protein stability of the pro-apoptotic protein Bax.

Project description:Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 degrees C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed.

| S-EPMC2778690 | biostudies-literature

Cdk2 phosphorylation of Bcl-xL after stress converts it to a pro-apoptotic protein mimicking Bax/Bak.

Project description:Apoptosis is a regulated form of cell death that proceeds by defined biochemical pathways. Most apoptosis is controlled by interactions between pro-survival and pro-apoptotic Bcl-2 family proteins in which death is often the consequence of permeabilization of the mitochondrial outer membrane. Many drugs affect this equilibrium to favor apoptosis but this process is not completely understood. We show that the chemotherapeutic drug cisplatin initiates an apoptotic pathway by phosphorylation of a pro-survival Bcl-2 family member, Bcl-xL, by cyclin-dependent kinase 2. The phosphorylation occurred at a previously unreported site and its biologic significance was demonstrated by a phosphomimetic modification of Bcl-xL that was able to induce apoptosis without addition of cisplatin. The mechanism of cell death induction was similar to that initiated by pro-apoptotic Bcl-2 family proteins, that is, phosphorylated Bcl-xL translocated to the mitochondrial membrane, and formed pores in the membrane. This initiated cytochrome c release and caspase activation that resulted in cell death.

| S-EPMC4877050 | biostudies-literature

Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers.

Project description:The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices ?1 and ?6 disengage from the rest of the domain, leaving helices ?2-?5 as a folded unit. Helices ?2-?5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX "BH3-in-groove homodimer." Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of ?3 and ?5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a "worm hole."

| S-EPMC3908389 | biostudies-literature

miR-128 exerts pro-apoptotic effect in a p53 transcription-dependent and -independent manner via PUMA-Bak axis.

Project description:p53 has attracted tremendous attention due to its master role in tumor development. Activation of p53 in tumor cells has been the prime focus for cancer drug discovery. Recent studies have shown that few miRNAs can regulate p53 activity directly or indirectly. We herein demonstrate that miR-128 positively regulates p53 activity. Our data suggest that miR-128 inhibits SIRT1 expression directly through a miR-128-binding site within the 3' UTR of SIRT1. miR-128 inhibition of SIRT1 led to an increase in acetylated p53 and its transcriptional targets. miR-128 decreased phospho-Akt and phospho-FOXO3A, increased acetylated FOXO3A and promoted FOXO3A translocation to the nucleus. We further demonstrated that miR-128 augments the antitumor effect of compounds that target the p53 pathway. Furthermore, miR-128 induces apoptosis in wild (WT) p53 as well as in mutant p53-expressing cells in a p53-dependent and -independent manner via induction of PUMA. Pretreatment with PUMA and Bak siRNAs abolished miR-128-induced apoptosis in HCT116 p53+/+ and HCT116 p53-/- cells. Taken together, we present the first evidence of miR-128 to be a new component joining the p53 network. This study emphasizes that miR-128 is a novel mitochondria-targeted miRNA that can be further evaluated as a chemotherapeutic agent for human cancers as it induces apoptosis irrespective of p53 status.

| S-EPMC3613825 | biostudies-literature

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