Crystal structure of Bax bound to the BH3 peptide of Bim identifies important contacts for interaction

The BH3-only protein Bim is a potent direct activator of the proapoptotic effector protein Bax, but the structural basis for its activity has remained poorly defined. Here we describe the crystal structure of the BimBH3 peptide bound to BaxΔC26 and structure-based mutagenesis studies. Similar to BidBH3, the BimBH3 peptide binds into the cognate surface groove of Bax using the conserved hydrophobic BH3 residues h1–h4. However, the structure and mutagenesis data show that Bim is less reliant compared with Bid on its ‘h0'' residues for activating Bax and that a single amino-acid difference between Bim and Bid encodes a fivefold difference in Bax-binding potency. Similar to the structures of BidBH3 and BaxBH3 bound to BaxΔC21, the structure of the BimBH3 complex with BaxΔC displays a cavity surrounded by Bax α1, α2, α5 and α8. Our results are consistent with a model in which binding of an activator BH3 domain to the Bax groove initiates separation of its core (α2–α5) and latch (α6–α8) domains, enabling its subsequent dimerisation and the permeabilisation of the mitochondrial outer membrane.The intrinsic pathway to apoptosis is regulated by interactions between members of three factions of the Bcl-2 protein family: the BH3-only proteins such as Bim and Bid, which initiate the process, the essential effectors Bax and Bak, and the prosurvival members, which oppose the action of both other factions.¹ The interactions between prosurvival Bcl-2 family members and BH3 peptides have been well characterised as the earliest studies with Bcl-x_L and a BakBH3 peptide.² Such complexes are readily formed in solution by incubating the C-terminally (ΔC) truncated prosurvival Bcl-2 protein with a BH3 peptide. The absence of the C-terminal segment that can anchor the Bcl-2 protein in a membrane apparently has little effect on the ensuing complex. That complex is believed to be responsible for the antiapoptotic function of Bcl-2, by sequestration of the BH3 motif either of the so-called BH3-only proteins such as Bim (''mode 1'') or of Bax or Bak (''mode 2'').³Although proapoptotic Bax and Bak have very similar three-dimensional structures to their prosurvival relatives,^{4, 5, 6} until recently^{7, 8} no structure of a complex of either Bax or Bak with a BH3 peptide had been captured, despite an accumulation of evidence that Bax and Bak could be activated directly by interaction with the BH3-only proteins Bid, Bim and possibly others.^{9, 10, 11, 12, 13}Unlike Bak, which is constitutively anchored in the mitochondrial outer membrane (MOM) via its C-terminal segment, Bax is largely cytosolic in healthy cells and accumulates at the MOM only upon a death signal.^{14, 15} There it is believed to display at least two different conformers,^{16, 17} one loosely associated with the MOM and another in which its membrane anchor (helix α9) is inserted into the MOM. In striking contrast to the antiapoptotic relatives of Bcl-2, a construct of Bax lacking its C-terminal membrane anchor, BaxΔC21, has no measurable interaction with BH3 peptides. However, in the presence of the detergent octylglucoside binding is detected by surface plasmon resonance (SPR) for the BH3 peptides of Bim, Bid, Bak and Bax itself with IC50s in the range of 0.1–1μM,^{7, 18} some 100-fold weaker compared with those measured similarly with (for example) Bcl-x_LΔC, where no detergent is required. Weaker interactions between BidBH3 or BimBH3 and BaxΔC as compared with Bcl-x_LΔC are not inconsistent with various models for the function of the Bcl-2 protein family whereby the prosurvival molecules sequester BH3 motifs with high affinity and long half-lives, but proapoptotic Bax and Bak are activated by transient (‘hit-and-run'') interactions with BH3 motifs.^{19, 20, 21}Complexes of BaxΔC21 bound to BH3 peptides from Bid and Bax have been prepared by coincubation of the protein with CHAPS and an excess of the peptides.⁷ Under these conditions, the protein undergoes a conformational change and dimerises via domain swapping of helical segments α2–α5 and α6–α8, dubbed ‘core'' and ‘latch'' domains, respectively. Although this ‘core/latch dimer'' is thought to be an in vitro artefact, its formation is diagnostic for the core and latch separation, which is required for membrane-associated Bax to dimerise via its core domains and then to permeabilise the MOM.⁷ If the latch domain is absent, as in a recombinant construct of GFP fused to Bax α2–α5, the core domain forms BH3:groove symmetric dimers,⁷ which, consistent with a wide body of evidence,^{21, 22, 23, 24, 25} are present in apoptotic pores.Previous work⁷ highlighted the importance of two hydrophobic ‘h0'' residues (Figure 1) in the peptide (I82/I83 in BidBH3) in governing Bid''s ability to activate Bax. Similar to Bid, Bim is also a potent direct activator of Bax, and the ‘h0'' amino acids in Bim are proline and glutamic acid. In the absence of a structure of BimBH3:BaxΔC, it remained unclear how these ‘h0'' residues were accommodated. Here we describe the crystal structures of BimBH3 26- and 20-mer peptides bound to BaxΔC26. Comparison with the structure of BidBH3:BaxΔC21 allows a dissection of the critical contacts between these two peptides and BaxΔC. The binding profiles of mutant BH3 peptides illustrate that BimBH3 binding to Bax is less dependent on the ‘h0'' residues compare with that in the case for BidBH3. The BimBH3 complex displays a similar cavity adjacent to Bax α1, α2, α5 and α8 as seen in the BidBH3 complex. We also describe a structure of BidBH3 bound to a BaxΔC21 mutant, I66A, which is more typical of the BH3 signature of antiapoptotic Bcl-2 family proteins^{7, 26}Open in a separate windowFigure 1BimBH3 binds BaxΔC. (a) BH3 peptide sequences used in this study, indicating the 5 hydrophobic amino-acid positions ‘h0''–‘h4''. (b) The core/latch dimer of BaxΔC26 bound to BimBH3. The two Bax polypeptides, shown here as cartoons, are coloured yellow and grey, and the two Bim peptides cyan and orange. A crystallographic dyad symmetry axis passes through the centre of this particle. (c) Structure of BimBH3:BaxΔC26 complex. The globular unit depicted comprises Bax residues 1–128 from one polypeptide and 129–166 from the other, together with the associated Bim peptide. Bax is represented by its surface and colour coded according to surface charge (blue, positive potential (4kT/e); red, negative potential (−2kT/e); calculated using the Adaptive Poisson–Boltzmann Solver.⁴¹ The trace of the Bim peptide (cyan) is shown with ‘h0'' (P144, E145), ‘h1'' (I148), ‘h2'' (L152), ‘h3'' (I155) and ‘h4'' (F159) represented as sticks. (d) Overlay of BimBH3:BaxΔC26 with BidBH3:BaxΔC21 (PDB:4BD2). Structures represented as cartoon ribbons, yellow for Bax in the Bim complex and magenta for Bax in the Bid complex. The peptides (Bim cyan and Bid blue) stand vertically in the foreground in this view (similar to Figure 1c), with their N termini at the bottom of the figure