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

Abstract

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.

Figure 1.

Structural similarity between Bcl-x_L (left, from 1MAZ.pdb) and the translocation domain of diphtheria toxin (right, from 1DDT.pdb). The hydrophobic helical hairpin motifs, helices 5,6 of Bcl-x_L and helices 8,9 of diphtheria toxin T-domain are represented by a darker shade of gray. These figures were generated using MOLSCRIPT (Kraulis 1991).

Figure 2.

The thermodynamic stability of Bcl-x_LΔTM is slightly reduced upon acidification. The circular dichroic signal at 222 nm of Bcl-x_LΔTM was monitored as a function of GdnHCl and the resulting data fit as described in the text. The free energy of folding, ΔG° (H₂O), under conditions that do not favor membrane insertion (pH 7.4 [•]) (A) is 15.8 ± 1.2 kcal · mol⁻¹. Under conditions that favor membrane insertion (pH 4.9 [○]) (B), ΔG° (H₂O) is 14.6 ± 1.0 kcal·mol⁻¹. The m_G value decreases from 4.3 ± 0.3 kcal·mol⁻¹·M⁻¹ at pH 7.4 to 3.5 ± 0.2 kcal · mol⁻¹ · M⁻¹ at pH 4.9.

Figure 3.

Bcl-x_LΔTM is monomeric in solution upon acidification. Representative sedimentation equilibrium data are presented for 25 μM Bcl-x_LΔTM at a XLI rotor speed of 19,000 rpm in solution at 25°C collected at pH 7.4 (A) and 4.9 (B). The data are well described by fitting to a single species (—) that is the molecular weight of monomeric Bcl-x_LΔTM within experimental uncertainty. Every second data point is displayed (○) but all data were globally fit. The fit to the molecular weight of the dimer of Bcl-x_LΔTM (- - -) and the residuals to the fits are also displayed. The global fit of the data from three concentrations and XLI rotor speeds of 17,000, 19,000, and 22,000 rpm was carried out using a modified Lamm-Svedberg equation as described in the text. The global fit to six data sets is presented as supplemental data.

Figure 4.

Secondary structure of Bcl-x_LΔTM is conserved upon acidification. The far-UV circular dichroism spectra were collected at pH 7.4 (•) and pH 4.9 (○).

Figure 5.

The backbone structure of Bcl-x_LΔTM does not undergo significant conformational change upon acidification. ¹H-¹⁵N HSQC spectra of Bcl-x_LΔTM were collected on a 0.6-mM sample of uniformly ¹⁵N labeled protein as a function of pH. (A) A representative ¹H-¹⁵N HSQC spectrum for Bcl-x_LΔTM at pH 7.4 is shown. The differences in amide proton chemical shift between pH 7.4 and pH 5.9 (B), pH 5.3 (C), and pH 4.9 (D) are displayed. Note that some minor differences are expected due to the salt dependence of the chemical shift (Schaller and Robertson 1995). Differences in amide nitrogen chemical shift also showed no significant changes as a function of pH (data not shown).

Figure 6.

The integrity of the hydrophobic core packing in Bcl-x_LΔTM is conserved upon acidification. Near-UV spectra were collected at pH 7.4 (•) and pH 4.9 (○).

Figure 7.

No indication of molten–globule formation upon acidification of Bcl-x_LΔTM. ¹H-¹³C HSQC spectra of Bcl-x_LΔTM were collected on a 0.8-mM unlabeled protein sample (natural abundance ¹³C) as a function of pH. Data shown only for pH 7.4 (A) and pH 4.9 (B).