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. 2009 Oct 30;284(44):30508-17.
doi: 10.1074/jbc.M109.040725. Epub 2009 Sep 2.

Conformational changes in Bcl-2 pro-survival proteins determine their capacity to bind ligands

Erinna F Lee  1 , Peter E Czabotar, Hong Yang, Brad E Sleebs, Guillaume Lessene, Peter M Colman, Brian J Smith, W Douglas Fairlie
Affiliations

Conformational changes in Bcl-2 pro-survival proteins determine their capacity to bind ligands

Erinna F Lee et al. J Biol Chem. .

Abstract

Antagonists of anti-apoptotic Bcl-2 family members hold promise as cancer therapeutics. Apoptosis is triggered when a peptide containing a BH3 motif or a small molecule BH3 peptidomimetic, such as ABT 737, binds to the relevant Bcl-2 family members. ABT-737 is an antagonist of Bcl-2, Bcl-x(L), and Bcl-w but not of Mcl-1. Here we describe new structures of mutant BH3 peptides bound to Bcl-x(L) and Mcl-1. These structures suggested a rationale for the failure of ABT-737 to bind Mcl-1, but a designed variant of ABT-737 failed to acquire binding affinity for Mcl-1. Rather, it was selective for Bcl-x(L), a result attributable in part to significant backbone refolding and movements of helical segments in its ligand binding site. To date there are few reported crystal structures of organic ligands in complex with their pro-survival protein targets. Our structure of this new organic ligand provided insights into the structural transitions that occur within the BH3 binding groove, highlighting significant differences in the structural properties of members of the Bcl-2 pro-survival protein family. Such differences are likely to influence and be important in the quest for compounds capable of selectively antagonizing the different family members.

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Figures

FIGURE 1.
FIGURE 1.
Structure of BimL12Y·Mcl-1 complex. a, comparison of Leu-12 and L12Y binding pockets on wild-type (PDB 2NL9) and mutant Bim BH3 peptides bound to Mcl-1. The tyrosine at position 12 in the Bim mutant peptide (lower panel) penetrates the h2 binding pocket deeper than the wild-type leucine residue. A water molecule (indicated in red) is seen at the bottom of the pocket in both structures. b, overlay of Mcl-1 and Bcl-xL structures in their respective complexes with BimL12Y (teal) and ABT-737 (yellow, PDB 2YXJ), showing only the peptide and organic ligand. The tyrosine in BimL12Y penetrates the h2 pocket to approximately the same depth that the chlorobiphenyl group of ABT-737 penetrates the corresponding pocket of Bcl-xL; however, the angle of entry (arrows) is different.
FIGURE 2.
FIGURE 2.
A designed ABT-737 derivative, W1191542. a, chemical structures of ABT-737, ABT-263, Compound 43b (23) and W1191542. b, the IC50 values (in nm) determined by solution competition assays for either ABT-737 or W1191542 binding mammalian pro-survival proteins. ABT-737 binds tightly to Bcl-xL, Bcl-w, and Bcl-2, whereas W1191542 demonstrates a more selective binding profile for Bcl-xL.
FIGURE 3.
FIGURE 3.
Biological functioning of W1191542. a, W1191542 elicits cytochrome c release from digitonin-permeabilized mcl-1−/− MEFs, similar to ABT-737 and Bad BH3 domain peptide but not bcl-x−/− MEFs, evidenced by its translocation into the soluble fraction. WB, Western blot. b and c, W1191542 kills MEFs deficient in Mcl-1 in short-term (24 h) (b) viability and long term (6 days) clonogenic assays (c) but is significantly less efficacious than ABT-737. Crosses indicate the absence of colony formation. d, binding kinetics of ABT-737 and W1191542 to Bcl-xL. W1191542 binds Bcl-xL more rapidly but dissociates faster than ABT-737.
FIGURE 4.
FIGURE 4.
Structure of the W1191542·Bcl-xL complex. a, overlay of structures of Bcl-xL in complex with ABT-737 (Bcl-xL, orange; ABT-737, yellow), W1191542 molecule A (Bcl-xL, green; W1191542/A, pink), or W1191542 molecule B (Bcl-xL, blue; W1191542/B, gray). Note the significant differences in the orientation of the α3 and α4 helices. b, close-up view of each compound in the region of the h2 pocket. The biphenyl of W1191542 molecule A projects out of the binding groove located between the α3 and α4 helices, whereas in W1191542 molecule B and ABT-737 it projects into the groove, although their orientations are different. c, overlay of the structures of Bcl-xL in complex with Compound 43b (Bcl-xL, orange; Compound 43b, yellow) (23), W1191542 molecule A (Bcl-xL, green; W1191542/A, pink), or W1191542 molecule B (Bcl-xL, blue; W1191542/B, gray). The space occupied by the biphenyl of Compound 43b overlays with that used by the biphenyl moiety in W1191542 molecule B.
FIGURE 5.
FIGURE 5.
Structure of the BimL12F·Bcl-xL complex. a, overlay of structures of Bcl-xL in complex with wild-type Bim BH3 (Bcl-xL, pink; Bim BH3, gray) or BimL12F (Bcl-xL, cyan; BimL12F, dark blue). b, close-up view in the region of the h2-binding pocket showing the relative orientation of the wild-type leucine residue in Bim BH3 (gray) or the phenylalanine residue at the same position in the mutated peptide (dark blue). c, overlay of the structures of Bcl-xL in complex with BimL12F (Bcl-xL, cyan; BimL12F, dark blue), ABT-737 (Bcl-xL, orange; ABT-737, yellow), or W1191542 molecule B (Bcl-xL, light blue; W1191542/A, gray). The phenylalanine side chain projects into the h2 binding pocket similarly to the chlorobiphenyl moiety of ABT-737.
FIGURE 6.
FIGURE 6.
Proposed structural transitions in the binding of W1151952 to Bcl-xL. a, the lower half of the binding groove (α3 helix, pink; α4 helix, blue) is occluded in unliganded Bcl-xL (PDB 1MAZ). b, upon engaging W1191542, there is a partial opening of the groove to accommodate the conformer observed in molecule A. c, when the biphenyl moiety penetrates the h2 pocket, as in molecule B, the groove is further widened.
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