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Evidence that membrane insertion of the cytosolic domain of Bcl-xL is governed by an electrostatic mechanism.

ABSTRACT: Signals from different cellular networks are integrated at the mitochondria in the regulation of apoptosis. This integration is controlled by the Bcl-2 proteins, many of which change localization from the cytosol to the mitochondrial outer membrane in this regulation. For Bcl-xL, this change in localization reflects the ability to undergo a conformational change from a solution to integral membrane conformation. To characterize this conformational change, structural and thermodynamic measurements were performed in the absence and presence of lipid vesicles with Bcl-xL. A pH-dependent model is proposed for the solution to membrane conformational change that consists of three stable conformations: a solution conformation, a conformation similar to the solution conformation but anchored to the membrane by its C-terminal transmembrane domain, and a membrane conformation that is fully associated with the membrane. This model predicts that the solution to membrane conformational change is independent of the C-terminal transmembrane domain, which is experimentally demonstrated. The conformational change is associated with changes in secondary and, especially, tertiary structure of the protein, as measured by far and near-UV circular dichroism spectroscopy, respectively. Membrane insertion was distinguished from peripheral association with the membrane by quenching of intrinsic tryptophan fluorescence by acrylamide and brominated lipids. For the cytosolic domain, the free energy of insertion (DeltaG degrees x) into lipid vesicles was determined to be -6.5 kcal mol(-1) at pH 4.9 by vesicle binding experiments. To test whether electrostatic interactions were significant to this process, the salt dependence of this conformational change was measured and analyzed in terms of Gouy-Chapman theory to estimate an electrostatic contribution of DeltaG degrees el approximately -2.5 kcal mol(-1) and a non-electrostatic contribution of DeltaG degrees nel approximately -4.0 kcal mol(-1) to the free energy of insertion, DeltaG degrees x. Calcium, which blocks ion channel activity of Bcl-xL, did not affect the solution to membrane conformational change more than predicted by these electrostatic considerations. The lipid cardiolipin, that is enriched at mitochondrial contact sites and reported to be important for the localization of Bcl-2 proteins, did not affect the solution to membrane conformational change of the cytosolic domain, suggesting that this lipid is not involved in the localization of Bcl-xL in vivo. Collectively, these data suggest the solution to membrane conformational change is controlled by an electrostatic mechanism. Given the distinct biological activities of these conformations, the possibility that this conformational change might be a regulatory checkpoint for apoptosis is discussed.

SUBMITTER: Thuduppathy GR

PROVIDER: S-EPMC1785297 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Evidence that membrane insertion of the cytosolic domain of Bcl-xL is governed by an electrostatic mechanism.

Thuduppathy Guruvasuthevan R GR Craig Jeffrey W JW Kholodenko Victoria V Schon Arne A Hill R Blake RB

Journal of molecular biology 20060406 4

Signals from different cellular networks are integrated at the mitochondria in the regulation of apoptosis. This integration is controlled by the Bcl-2 proteins, many of which change localization from the cytosol to the mitochondrial outer membrane in this regulation. For Bcl-xL, this change in localization reflects the ability to undergo a conformational change from a solution to integral membrane conformation. To characterize this conformational change, structural and thermodynamic measurement ...[more]

PMID: 16650855

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Project description:ABT-737 is a pharmacological inhibitor of the anti-apoptotic activity of B-cell lymphoma-extra large (Bcl-xL) protein; it promotes apoptosis of cancer cells by occupying the BH3-binding pocket. We have shown previously that ABT-737 lowers cell metabolic efficiency by inhibiting ATP synthase activity. However, we also found that ABT-737 protects rodent brain from ischemic injury in vivo by inhibiting formation of the pro-apoptotic, cleaved form of Bcl-xL, ΔN-Bcl-xL. We now report that a high concentration of ABT-737 (1 μM), or a more selective Bcl-xL inhibitor WEHI-539 (5 μM) enhances glutamate-induced neurotoxicity while a low concentration of ABT-737 (10 nM) or WEHI-539 (10 nM) is neuroprotective. High ABT-737 markedly increased ΔN-Bcl-xL formation, aggravated glutamate-induced death and resulted in the loss of mitochondrial membrane potential and decline in ATP production. Although the usual cause of death by ABT-737 is thought to be related to activation of Bax at the outer mitochondrial membrane due to sequestration of Bcl-xL, we now find that low ABT-737 not only prevents Bax activation, but it also inhibits the decline in mitochondrial potential produced by glutamate toxicity or by direct application of ΔN-Bcl-xL to mitochondria. Loss of mitochondrial inner membrane potential is also prevented by cyclosporine A, implicating the mitochondrial permeability transition pore in death aggravated by ΔN-Bcl-xL. In keeping with this, we find that glutamate/ΔN-Bcl-xL-induced neuronal death is attenuated by depletion of the ATP synthase c-subunit. C-subunit depletion prevented depolarization of mitochondrial membranes in ΔN-Bcl-xL expressing cells and substantially prevented the morphological change in neurites associated with glutamate/ΔN-Bcl-xL insult. Our findings suggest that low ABT-737 or WEHI-539 promotes survival during glutamate toxicity by preventing the effect of ΔN-Bcl-xL on mitochondrial inner membrane depolarization, highlighting ΔN-Bcl-xL as an important therapeutic target in injured brain.

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Evidence that membrane insertion of the cytosolic domain of Bcl-xL is governed by an electrostatic mechanism.

Publications

Evidence that membrane insertion of the cytosolic domain of Bcl-xL is governed by an electrostatic mechanism.

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