Ligand binding and membrane insertion compete with oligomerization of the BclXL apoptotic repressor

Abstract

B-cell lymphoma extra large (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 (1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine) 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.

Figure 1

BclXL domain organization and BH3 ligands. (a) Human BclXL is comprised of the BH4-BH3-BH1-BH2-TM modular organization, with a C-terminal transmembrane (TM) domain preceded by four N-terminal Bcl2 homology (BH) domains. The relationship between the various helices (α1–α9) punctuating the topological fold of BclXL and the BH domains is clearly indicated for clarity. While the BclXL_FL construct represents the full-length protein with the above modular organization, the C-terminal TM domain has been deleted in BclXL_dTM construct in order to investigate its function in the biological function of BclXL in this study. The numerals indicate amino acid boundaries within corresponding protein sequences. (b) Amino acid sequence alignment of 20-mer peptides spanning various BH3 domains within human Bid, Bad and Bax proteins. The numerals indicate amino acid boundaries within corresponding protein sequences. The LXXXXD motif characteristic of all BH3 domains is highlighted.

Figure 2

ITC analysis for the binding of Bid_BH3 peptide to BclXL_FL (a) and BclXL_dTM (b) constructs. The upper panels show raw ITC data expressed as change in thermal power with respect to time over the period of titration. In the lower panels, change in molar heat is expressed as a function of molar ratio of Bid_BH3 peptide to the corresponding construct. The solid lines in the lower panels show the fit of data to a one-site model, as embodied in Eq [1], using the ORIGIN software. The insets show same titrations conducted in the presence of DMPC/DHPC bicelles.

Figure 3

ALS analysis for BclXL_dTM and BclXL_FL constructs as indicated. (a) Elution profiles as monitored by the differential refractive index (Δn) plotted as a function of elution volume (V) for BclXL_FL (top panel) and BclXL_dTM (bottom panel) constructs. Note that the elution profile for BclXL_FL construct is shown at both 50μM (black) and 10μM (red) initial protein concentrations loaded onto the Superdex-200 column, while that for BclXL_dTM construct is only shown at 50μM (black). (b) Partial Zimm plots obtained from analytical SLS measurements at a specific protein concentration for BclXL_FL polymer (top panel) and BclXL_dTM monomer (bottom panel). The solid lines through the data points represent linear fits. (c) Autocorrelation function plots obtained from analytical DLS measurements at a specific protein concentration for BclXL_FL polymer (top panel) and BclXL_dTM monomer (bottom panel). The solid lines through the data points represent non-linear least squares fits to Eq [11].

Figure 4

TEM micrographs of negatively-stained BclXL_FL construct alone (a) and in the presence of Bid_BH3 peptide (b).

Figure 5

DSC isotherms for BclXL_FL construct at 50μM (a), BclXL_dTM construct at 50μM (b) and BclXL_FL construct at 10μM (c) alone (black), in the presence of excess Bid_BH3 peptide (red) and in the presence of excess DMPC/DHPC bicelles (green). The dashed vertical lines indicate T_m values of various thermal phases.

Figure 6

SSF spectra of BclXL_FL construct at 5μM (a), BclXL_dTM construct at 5μM (b) and SEC-resolved fractions containing higher-order oligomers of BclXL_FL at 1μM (c) alone (black), in the presence of excess Bid_BH3 peptide (red) and in the presence of excess DMPC/DHPC bicelles (green).

Figure 7

Far-UV CD spectra of BclXL_FL construct at 5μM (a), BclXL_dTM construct at 5μM (b) and SEC-resolved fractions containing higher-order oligomers of BclXL_FL at 1μM (c) alone (black), in the presence of excess Bid_BH3 peptide (red) and in the presence of excess DMPC/DHPC bicelles (green).

Figure 8

Structural models of full-length BclXL in three distinct conformations with respect to the C-terminal TM domain (α9 helix). (a) Monomeric BclXL with the TM domain exposed to solution (BclXL_solTM). (b) Monomeric BclXL with the TM domain bound to the canonical hydrophobic groove (BclXL_cisTM). (c) Homodimeric BclXL with the TM domain bound to the canonical hydrophobic groove but swapped in an intermolecular trans-fashion — the TM domain of one monomer (green) is bound to the other monomer (blue) and vice versa (BclXL_transTM).

Figure 9

MD analysis on structural models of full-length BclXL in three distinct conformations with respect to the C-terminal TM domain (α9 helix). (a) Root mean square deviation (RMSD) of backbone atoms (N, Cα and C) for residues 1–233 (black), residues 86–195 (red), residues 1–85 (green) and residues 196–233 (blue) within each simulated structure relative to the initial modeled structure of BclXL_solTM, BclXL_cisTM and BclXL_transTM as a function of simulation time. Note that, for each construct, the RMSD of full-length (FL) protein spanning residues 1–233 is also deconvoluted into the central core (CC) region spanning residues 86–195, the N-terminal (NT) region spanning residues 1–85, and the C-terminal (CT) region spanning residues 196–233. (b) Root mean square fluctuation (RMSF) of backbone atoms (N, Cα and C) averaged over the entire course of corresponding MD trajectory of BclXL_solTM, BclXL_cisTM and BclXL_transTM as a function of residue number.

Figure 10

Models for BclXL oligomerization and its role in apoptotic regulation. (a) Oligomerization of BclXL via a domain-swapped mechanism. The TM domain of one monomer (green) occupies the canonical hydrophobic groove within another monomer (blue) and vice versa to form a homodimer. The resulting homodimers, due to greater interacting molecular surface area, further self-associate into higher-order oligomers. (b) Oligomerization of BclXL via an inter-locking mechanism. The TM domain of one monomer (green) occupies the canonical hydrophobic groove within another monomer (blue) in a head-to-tail fashion so as to aid the assembly of much larger oligomers. (c) A thermodynamic cycle depicting how various linked-equilibria determine the fate of BclXL repressor to self-associate into higher-order [BclXL]_n oligomers versus hetero-association with activator (A) and effector (E) molecules in quiescent versus apoptotic cells. In quiescent non-apoptotic cells, BclXL either self-associates into higher-order [BclXL]_n oligomers and/or hetero-associates with effectors such as Bax and Bak, depending on the relative ratio of their cellular concentrations, to form BclXL-E repressor-effector complexes. In this manner, self-association into higher-order oligomers leads to inactivation of BclXL and hetero-association inactivates effectors. Upon receiving apoptotic stimuli, activators such as Bid and Bad compete with self-association of BclXL into higher-order oligomers and its hetero-association with effectors, leading to the formation of BclXL-A repressor-activator complexes as well as freeing up the effectors, which subsequently insert into MOM. This results in mitochondrial permeabilization leading to the release of apoptogenic factors that in turn induce cells to undergo apoptosis.