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Structure of Bcl-xL-Bak Peptide Complex: Recognition Between Regulators of Apoptosis

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Science  14 Feb 1997:
Vol. 275, Issue 5302, pp. 983-986
DOI: 10.1126/science.275.5302.983

Abstract

Heterodimerization between members of the Bcl-2 family of proteins is a key event in the regulation of programmed cell death. The molecular basis for heterodimer formation was investigated by determination of the solution structure of a complex between the survival protein Bcl-xL and the death-promoting region of the Bcl-2-related protein Bak. The structure and binding affinities of mutant Bak peptides indicate that the Bak peptide adopts an amphipathic α helix that interacts with Bcl-xL through hydrophobic and electrostatic interactions. Mutations in full-length Bak that disrupt either type of interaction inhibit the ability of Bak to heterodimerize with Bcl-xL.

Programmed cell death (apoptosis) occurs during the course of several physiological processes, and when dysregulated contributes to many diseases, including cancer, autoimmunity, and neurodegenerative disorders (1). The Bcl-2 family of proteins plays a central role in the regulation of apoptotic cell death induced by a wide variety of stimuli (2). Some proteins within this family, including Bcl-2 and Bcl-xL, inhibit programmed cell death, and others, such as Bax and Bak, can promote apoptosis. Interactions between these two groups of proteins antagonize their different functions and modulate the sensitivity of a cell to apoptosis (3, 4). Several regions of the death-inhibiting proteins participate in their antiapoptotic activity and heterodimerization with the death-promoting proteins, including the Bcl-2 homology 1 (BH1) and BH2 regions (3, 5, 6). In contrast, only a relatively small portion of the death-promoting proteins encompassing the BH3 region is critical for the ability to promote apoptosis (710). For example, small, truncated forms of Bak are necessary and sufficient both for promoting cell death and binding to Bcl-xL (7).

The three-dimensional (3D) structure of the cell survival protein Bcl-xL consists of two central hydrophobic α helices surrounded by five amphipathic helices (11). To understand how Bak interacts with Bcl-xL and inhibits the ability of Bcl-xL to promote cell survival, we determined the solution structure of Bcl-xL complexed with a 16-residue peptide derived from the BH3 region of Bak. We also measured the binding affinities of Bcl-xL to alanine mutant Bak peptides and to peptides corresponding to the BH3 regions of other Bcl-2 family members (12, 13).

The minimal region of Bak required to bind to Bcl-xL was examined in a fluorescence-based assay (14). A 16-amino acid peptide derived from the BH3 region of Bak (residues 72 to 87) bound tightly to Bcl-xL (Table 1). In contrast, smaller peptides from this region, such as an 11-amino acid peptide corresponding to residues 77 to 87, did not bind (Table 1). The 16-amino acid peptide of Bak corresponds precisely to the region of Bcl-xL that forms the second α helix (11).

Table 1.

Binding affinities (14) of peptides to Bcl-xL. Residues of Bak peptide substituted with alanine are in boldface. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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The structure of the 16-amino acid peptide (15) complexed with a biologically active deletion mutant of Bcl-xL (16) was determined by nuclear magnetic resonance spectroscopy (NMR). The structure was determined from a total of 2813 NMR-derived restraints and is well defined by the NMR data (Fig. 1A) (17). The atomic root-mean- square deviation (rmsd) about the mean coordinate positions for residues 1 to 205 of Bcl-xL and 72 to 87 of the Bak peptide was 0.79 ± 0.15 Å for the backbone and 1.21 ± 0.13 Å for all heavy atoms.

Fig. 1.

(A) Stereoview of the backbone (N, Cα, C′) of 15 superimposed NMR-derived structures of Bcl-xL (shown in black) complexed with the 16-amino acid Bak peptide (shown in red). (B) Ribbons (21) depiction of the averaged minimized NMR structure for the complex. The BH1, BH2, and BH3 regions of Bcl-xL are shown in yellow, red, and green, respectively. The Bak peptide is shown in magenta.

Overall, the structure of the truncated form of Bcl-xL when complexed to the Bak peptide is similar to the x-ray and NMR structures of uncomplexed Bcl-xL (11, 18). The Bak peptide binds in a hydrophobic cleft formed by the BH1, BH2, and BH3 regions of Bcl-xL (Figs. 1 and 2). Although a random coil when free in solution (19), the Bak peptide forms an α helix when complexed to Bcl-xL. The NH2-terminal residues of the peptide show numerous nuclear Overhauser effects (NOEs) to residues in the BH1 region of Bcl-xL (Val126, Glu129, Leu130, and Phe146), whereas the COOH-terminal portion of the Bak peptide interacts predominantly with residues in the BH2 and BH3 regions (Phe97, Arg100, Tyr101, and Phe105). The hydrophobic side chains of the peptide (Val74, Leu78, Ile81, and Ile85) point into a hydrophobic cleft of Bcl-xL (Fig. 2) and stabilize complex formation. In addition to these hydrophobic interactions, the charged side chains of the Bak peptide (Arg76, Asp83, and Asp84) are close to oppositely charged residues of Bcl-xL (Glu129, Arg139, and Arg100, respectively) (Fig. 2).

Fig. 2.

(A) Surface representation of the binding pocket of Bcl-xL bound to the Bak peptide. Hydrophobic residues showing NOEs to the peptide are colored in yellow, whereas Arg139/Arg100 and Glu129 are colored in blue and red, respectively. Residues of Bcl-xL are labeled in white and the Bak peptide in black. (B) Depiction of the side chains in the binding site of Bcl-xL. Hydrophobic side chains of the protein showing NOEs to the peptide are colored in yellow. Side chains of positively and negatively charged side chains interacting with the peptide are colored in blue and red, respectively. The peptide side chains are colored by atom type. Residues of Bcl-xL and the Bak peptide are labeled in black and green, respectively.

To identify the interactions that are important for complex formation, we measured the binding affinities of mutant Bak peptides containing alanine substitutions (Table 1) (14). A decrease in binding affinity by a factor of 800 was observed for the Bak peptide in which Leu78 is substituted by an alanine. This can be explained by the loss of extensive interactions between the side chain of Leu78 of Bak and the hydrophobic pocket formed by Tyr101, Leu108, Val126, and Phe146 of Bcl-xL (Fig. 2B). Mutation of other hydrophobic residues of Bak (Ile85, Ile81, and Val74) to alanine also resulted in reduced binding to Bcl-xL (Table 1), which further demonstrates the importance of hydrophobic interactions in complex formation. The hydrophobic residues at these positions are largely conserved in the Bcl-2 family of proteins (Table 1). In contrast, Ile80 is not conserved and is located on the surface of the complex (Fig. 2), consistent with the negligible loss in binding affinity observed when this residue was changed to an alanine.

Analysis of the structure (Fig. 2) suggested that the interaction between Asp83 of the Bak peptide and Arg139 of Bcl-xL would stabilize complex formation. Indeed, Asp83 is completely conserved within the Bcl-2 family of proteins, and when substituted with alanine in the Bak peptide, markedly reduced the binding of this peptide to Bcl-xL (Table 1). Moreover, Arg139 is highly conserved, and mutation of Arg139 to Gln in Bcl-xL inhibits its antiapoptotic activity and binding to the Bax protein (20). It was also expected from the structure (Fig. 2) that electrostatic interactions between Arg76 of Bak and Glu129 of Bcl-xL would contribute to complex formation. This is supported by the observed decrease in binding to Bcl-xL of a Bak peptide in which Arg76 is mutated to alanine (Table 1). However, the potential charge-charge interaction between Asp84 of Bak and Arg100 of Bcl-xL does not appear to be critical for complex stabilization as a negligible effect on binding to Bcl-xL was observed when Asp84 was substituted by an alanine (Table 1).

Interactions within the Bcl-2 family of proteins exhibit a defined selectivity and hierarchy (12, 13). To investigate whether this selectivity is conferred by the BH3 regions from other Bcl-2 family members, we measured the binding affinities of a series of BH3-containing peptides to Bcl-xL (14). Subtle differences in the amino acid sequences of the BH3 regions among members of the Bcl-2 family give rise to distinct differences in the affinities of these peptides for Bcl-xL (Table 1). The Bak peptide binds to Bcl-xL with greater affinity than any of the other peptides, including the peptides derived from the other death-promoting proteins, Bax and Bik. The Bcl-xL peptide binds with the weakest affinity to Bcl-xL, consistent with the monomeric nature of this protein (11). The selectivity of Bcl-xL that we observed for the peptides from different Bcl-2 family members is consistent with the selectivity for heterodimer formation amongst the Bcl-2 family of proteins and suggests that the BH3 region plays a central role in defining the binding specificity of the Bcl-2-related proteins for Bcl-xL.

The molecular interactions that stabilize the Bcl-xL-Bak peptide complex likely reflect the important interactions that occur between the full-length proteins. The wild-type Bak peptide can inhibit the interaction of Bcl-xL with full-length Bak or Bax in a concentration-dependent manner (20). Furthermore, Bak peptides containing alanine substitutions for Leu78 and Asp83, which markedly reduced their binding to Bcl-xL (Table 1), were unable to block heterodimer formation between-full length Bcl-xL and Bak (Fig. 3A). When these two residues (Leu78 and Asp83) were mutated in the full-length Bak protein, the mutant Bak proteins failed to coprecipitate with Bcl-xL even though they were expressed at levels comparable to that of the wild type protein (Fig. 3B). Thus, the reduction in binding to Bcl-xL observed with the full-length mutant Bak proteins resembles the loss in binding to Bcl-xL measured for the mutant Bak peptides. These data are consistent with previous reports (710) on the functional importance of the BH3 region of the death-promoting proteins. This region of Bak and similar sequences in Bax and Bik (Bip1) promote apoptosis and interact with Bcl-xL (7, 8). In addition, neither the BH1 nor the BH2 region of Bax is necessary for binding to Bcl-2 or for promoting cell death (9, 10).

Fig. 3.

(A) Mutations of critical residues in the Bak BH3 peptide abolish its ability to inhibit Bcl-xL heterodimerization with Bak. In vitro-translated Bcl-xL and Bak were combined together with 100 μM of the indicated Bak BH3 peptide. The reaction was immunoprecipitated with an antibody to Bcl-x (anti-Bcl-x), and the immunoprecipitated products were resolved by SDS-polyacrylamide gel electrophoresis (PAGE). (B) Mutations in Bak BH3 residues that are predicted to be involved in Bcl-xL-Bak interactions abolish heterodimerization. In vitro-translated Bcl-xL, Bak, or mutants of Bak were combined as indicated and immunoprecipitated with anti-Bcl-x. The immunoprecipitated products were resolved by SDS-PAGE. Bak mutation 1 contains a glutamic acid in place of arginine at amino acid 76 and an arginine in place of aspartic acid at amino acid 83. Bak mutant 2 contains an alanine in place of leucine at amino acid 78.

Using the structure of the Bcl-xL-Bak peptide complex and a homology model of the Bak protein, we modeled the structure of the heterodimer of the full-length proteins. In the structure of Bak based on its homology to Bcl-xL, the hydrophobic side chains of the amphipathic α2 helix containing the BH3 region point toward the interior of the Bak protein, making these residues unavailable to interact with Bcl-xL. Thus, binding to Bcl-xL would necessitate a conformational change in the Bak protein to expose the hydrophobic surface of α2. One possibility is a rotation of the α2 helix along the helix axis that would allow the formation of the same hydrophobic and charge-charge interactions observed in the NMR structure of the Bcl-xL-Bak peptide complex. It is of interest that based on the structure of Bcl-xL, this helix is predicted to be flanked by highly flexible loops on both ends that could allow such a rotation.

In summary, our structure of the Bcl-xL-Bak peptide complex reveals the structural basis for the requirements of the BH1, BH2, and BH3 regions for heterodimer formation among Bcl-2 family members. These data suggest that the formation of a hydrophobic binding cleft and properly positioned charged residues are required for the antiapoptotic functions of Bcl-xL. Indeed, a variety of mutations that would be predicted to alter the accessibility or binding properties of this region in Bcl-xL and Bcl-2, including G138A (3), R139Q (20), Y101K (20), and L130A (20), have been shown to inhibit the function of this protein. For proteins that promote cell death, only the BH3 region is required for activity (710), which as shown here forms an amphipathic α helix and binds with high affinity to the hydrophobic groove in Bcl-xL. Some proteins that promote cell death—such as Bik—have homology to other Bcl-2 proteins only within the BH3 region. In contrast, other Bcl-2-related proteins such as Bak or Bax are predicted to have more extensive structural similarities to Bcl-xL. For these proteins, our studies suggest that a structural change may be required for the BH3 region to participate in dimerization.

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