Ligand Binding and Membrane Insertion Compete with Oligomerization of the BclXL Apoptotic Repressor

Abstract

BclXL apoptotic repressor plays a central role in determining the fate of cells to live or die during physiological processes such as embryonic development and tissue homeostasis. Herein, using a myriad of biophysical techniques, we provide evidence that ligand binding and membrane insertion compete with oligomerization of BclXL in solution. Of particular importance is the observation that such oligomerization is driven by the intermolecular binding of its C-terminal transmembrane (TM) domain to the canonical hydrophobic groove in a domain-swapped trans-fashion, whereby the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa. Binding of BH3 ligands to the canonical hydrophobic groove displaces the TM domain in a competitive manner allowing BclXL to dissociate into monomers upon hetero-association. Remarkably, spontaneous insertion of BclXL into DMPC/DHPC bicelles results in a dramatic conformational change such that it can no longer recognize the BH3 ligands in what has come to be known as the “hit-and-run” mechanism. Collectively, our data suggest that oligomerization of a key apoptotic repressor serves as an allosteric switch that fine tunes its ligand binding and membrane insertion pertinent to the regulation of apoptotic machinery.

Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor

“…Full-length human BclXL (residues 1-233) was cloned into pET30 bacterial expression vector with an N-terminal His-tag using Novagen LIC technology, expressed in Escherichia coli BL21*(DE3) bacterial strain (Invitrogen) and purified on a Ni-NTA affinity column using standard procedures as described previously [40]. Protein concentration was determined by the fluorescence-based QuantIt assay (Invitrogen) and spectrophotometrically on the basis of an extinction coefficient of 47,440 M −1 cm −1 calculated for the full-length BclXL using the online software ProtParam at ExPasy Server [41].…”

“…We have previously shown that BclXL displays the propensity to oligomerize in solution and that such oligomerization is driven by the intermolecular binding of its C-terminal TM domain to the canonical hydrophobic groove in a domain-swapped trans-fashion, whereby the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa [40]. We postulated that such oligomerization serves as a regulatory switch to turn the anti-apoptotic action of BclXL “off” in quiescent cells but “on” in response to apoptotic cues.…”

Conformation of BCL-XL upon Membrane Integration

“…Treatment of BCL-XLΔC with basic pH, heat or detergent has also been shown to induce the formation of a domain-swapped symmetric homodimer, in which helices α6-α8 of one monomer participate in forming the prototypical globular structure of the other 23; 24; 25; 26 . At physiological pH in detergent-free solution, we did not observe dimer formation, even after raising the temperature to 45°C, as evidenced by size exclusion chromatography and the absence of characteristic NMR peak changes associated with conformational reorganization of the α5-α6 hinge.…”

“…Compared to their water-soluble states, membrane-bound BCL-XL and BCL-W have been proposed to display diminished affinity for their BH3 ligands due to a loosening of the groove structure in the hydrophobic environment 26; 33; 34 . According to current models, membrane binding by water-soluble BCL-2 proteins induces major conformational changes, while BH3 ligand binding by membrane-integrated BCL-2 proteins causes ulterior structural changes and ligand release 3 .…”

When detergent meets bilayer: Birth and coming of age of lipid bicelles

“…For example, details of membrane binding of myristoylated and non-myristoylated ARF1, ADP-ribosylation factor 1 (pdb-id 2K5U), was compared in isotropic bicelles [294]. Spontaneous insertion of the apoptotic repressor BclXL into q = 0.5 DMPC/DHPC bicelles was found to cause a dramatic conformational change that inhibits binding with its BH3 ligand [295]. Isotropic bicelles were used to supply lipid substrate to cobra venom phospholipase A 2 for kinetic assays [296].…”