The N-terminal domain of Bcl-xL reversibly binds membranes in a pH-dependent manner.

Bcl-xL regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-xLDeltaTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bcl-xLDeltaTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-xL activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-xL mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis.

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

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.

Acid destabilization of the solution conformation of Bcl-xL does not drive its pH-dependent insertion into membranes.

Regulation of programmed cell death by Bcl-xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH-dependence similar to the translocation domain of diphtheria toxin, where an acid-induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl-xL deltaTM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near- and far-UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl-xL deltaTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-xL deltaTM (deltaG(o)) was determined to be 15.8 kcal x mol(-1). Surprisingly, under conditions that favor a membrane conformation (pH 4.9), deltaG(o) was 14.6 kcal x mol(-1). These results differ from those obtained with many other membrane-insertable proteins where acid-induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl-xL and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl-xL from intracellular proteases as it undergoes this conformational change essential for its activity.

Designing protein dimerizers: the importance of ligand conformational equilibria.

In an effort to elucidate the role of ligand conformation in induced protein dimerization, we synthesized a flexible methotrexate (MTX) dimer, demonstrated its ability to selectively dimerize Escherichia coli dihydrofolate reductase (DHFR), and evaluated the factors regulating its ability to induce cooperative dimerization. Despite known entropic barriers, bis-MTX proved to possess substantial conformational stability in aqueous solution (-3.8 kcal/mol >/= DeltaG(fold) >/= -4.9 kcal/mol), exerting a dominant influence on the thermodynamics of dimerization. To dimerize DHFR, bis-MTX must shift from a folded to an extended conformation. From this conclusion, the strength of favorable protein-protein interactions in bis-MTX-E. coli DHFR dimers (-3.1 kcal/mol >/= DeltaG(c) >/= -4.2 kcal/mol), and the selectivity of dimerization for E. coli DHFR relative to mouse DHFR (>10(7)) could be determined. The crystal structure of bis-MTX in complex with E. coli DHFR confirms the feasibility of a close-packed dimerization interface and suggests a possible solution conformation for the induced protein dimers. Consequently, the secondary structure of this minimal foldamer regulates its ability to dimerize dihydrofolate reductase in solution, providing insight into the complex energy landscape of induced dimerization.