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.

The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner

“…In this assay, protein and lipid vesicles are incubated and then physically separated by centrifugation. For Bcl-x L ΔTM at pH 7.0 and 150 mM NaCl, we observe ~10% of the protein associated with lipid vesicles composed of DOPC and DOPG (60:40 and a 1:200 protein:lipid ratio), consistent with our earlier observations (19). When the pH of this sample was lowered to 4.0 via the addition of small amounts of concentrated HCl and resedimented, less than 5% of Bcl-x L ΔTM was detected in the supernatant, indicating nearly complete association with lipid vesicles (Figure 1).…”

“…These data allow us to refine our model for the conformational change of full-length Bcl-x L that involves at least three distinct conformations, one solution and two membrane (19). In this model, the solution conformation is similar to the known structure of Bcl-x L ΔTM (23), with the exception that the C-terminal transmembrane domain is tucked into its own BH3 binding pocket as in the structure of Bax, a pro-apoptotic Bcl-2 family member (52).…”

“…Using a construct lacking the transmembrane domain was essential for determining what drives membrane association of the N-terminal domain since full-length protein associates with lipid vesicles under conditions where Bcl-x L ΔTM does not (19). The critical surface pressure (π c ) of Bcl-x L ΔTM is consistent with membrane insertion at pH 5.0, but not at pH 7.0 (Figure 3a).…”

“…15 N-labeled Bcl-x L ΔTM was prepared as described previously (19 65 by the addition of small amounts of 0.1 N HCl. A HSQC spectrum was collected at this pH, and then the sample was titrated to pH 7.4 by the addition of small amounts of 0.1 N NaOH.…”

Amyloid Fibrils Formed by the Programmed Cell Death Regulator Bcl-xL

“…Moreover, the detection in cells of monomers, homodimers, and heterodimers as well as larger assemblies in the cytosol and in membranes suggests that Bcl-xL can adopt different supramolecular assemblies. [24][25][26][27][28]34,39,[44][45][46] These various states can lead to opposing effects of the protein during the regulation of apoptosis. 47,48 Yet, the functional implications of each form are poorly understood.…”