Redefining the BH3 Death Domain as a 'Short Linear Motif'

Abstract

B cell lymphoma-2 (BCL-2)-related proteins control programmed cell death through a complex network of protein-protein interactions mediated by BCL-2 homology 3 (BH3) domains. Given their roles as dynamic linchpins, the discovery of novel BH3-containing proteins has attracted considerable attention. However, without a clearly defined BH3 signature sequence the BCL-2 family has expanded to include a nebulous group of nonhomologous BH3-only proteins, now justified by an intriguing twist. We present evidence that BH3s from both ordered and disordered proteins represent a new class of short linear motifs (SLiMs) or molecular recognition features (MoRFs) and are diverse in their evolutionary histories. The implied corollaries are that BH3s have a broad phylogenetic distribution and could potentially bind to non-BCL-2-like structural domains with distinct functions.

Keywords: BCL-2 family; BH3; MoRF/MoRE; SLiM; globular domains; intrinsically disordered proteins.

Figure 1

B Cell Lymphoma-2 (BCL-2)-Related Proteins, BCL-2 Homology 3 (BH3)-Coding Exons, and BH3 Sequence Motif Logos. (A) Molecular interactions between BH3-only proteins and BCL-2-homologous proteins. BCL-2-related proteins fall into two categories, each with two subcategories: the proapoptotic BH3-only proteins, which are divided into activators (dark blue) and sensitizers (light blue), and the BCL-2-homologous proteins, which can be anti- or proapoptotic (light and dark purple, respectively). BH3-only protein localization and function are regulated at transcriptional, translational, and post-translational levels in response to specific death stimuli (reviewed in [6]). Activator BH3-only proteins can directly activate BAX or BAK by a transient physical interaction leading to insertion into mitochondrial membranes, oligomerization, mitochondrial outer membrane permeabilization (MOMP), and apoptosis. Sensitizer BH3-only proteins induce such phenomena only by docking their BH3 α helix into a hydrophobic groove on the surface of prosurvival BCL-2 homologs, thereby releasing BAX/BAK or activator BH3-only proteins , , . Although it behaves as a direct activator BH3-only protein, the BID protein appears to be a bona fide BCL-2/BAX homolog sharing a similar characteristic 3D fold (hence the two colors: half blue, half purple). All BH3-bearing proteins except BID are natively unfolded proteins (e.g., BIM, BMF) or belong to unrelated protein families having or predicted to have distinct structures. Depicted molecular interactions were extracted from Table S1 in the supplementary material online. (B) Logos resulting from the alignment of BH3 and BH3-like sequences of the various subgroups of BH3-containing proteins. Logo units are bits of information. Each position is displayed as a stack of amino acid letters with the height of the letter representing its proportion of the information content. The conserved Leu and Asp residues are visible in the BH3 core. Sequences are divergent outside this region. Stars indicate alignment gaps. Logos were calculated using Weblogo 3.4 (http://weblogo.threeplusone.com). Aligned sequences are the same as in Figure S1 in the supplementary material online (16 BCL-2-homologous proteins, eight canonical BH3-only proteins, 39 noncanonical BH3-bearing proteins). (C) Phase class of BH3-coding exons. The intron–exon gene structures of BAX and BAK are shown as examples. Exons (grey rectangles) are drawn to scale. The part of the exon encoding the BH3 motif is shown in black. Exons can be classified into nine different groups depending on the phases of their flanking introns: phase 0, reading frame interrupted between codons; phase 1, reading frame interrupted between the first and second nucleotides; phase 2, reading frame interrupted between the second and third nucleotides. See Figure S2 in the supplementary material online for complete intron–exon structures.

Figure I

Structure of the Antiapoptotic Protein B Cell Lymphoma (BCL)-xL in Complex with a 33-mer BIM BCL-2 Homology 3 (BH3) Peptide. The ribbon structure (PDB code 1PQ1[94]) shows that BCL-xL is an all-α-helical protein with a central hydrophobic core surrounded by amphipathic helices. This helical bundle arrangement is typical of anti- and proapoptotic BCL-2-homologous proteins. The BIM BH3 peptide (in blue) fits into an extended hydrophobic groove at the surface of BCL-xL. Various interactions (hydrophobic contacts, salt bridges, hydrogen bonds) are formed between BIM-BH3 residues and residues lining the BCL-xL groove. An aspartate residue (Asp) found in all BH3 motifs forms a salt bridge with a conserved arginine (Arg) residue present in the BH1 motif of BCL-xL (broken lines), and an invariant leucine (Leu) in the BIM-BH3 participates in hydrophobic contacts with the BH3-binding groove on the BCL-xL. The BH3 motif of BCL-xL is also colored blue. Diagrams of the two interacting proteins (right side) indicate the amino acid positions of BH3-containing helices and the positions of the nine BCL-xL α helices (numbered bars, light blue bars indicate helices that form the hydrophobic BH3-binding groove). The four BH motifs are shown in magenta (BH4), blue (BH3), yellow (BH1), and brown (BH2) and the C-terminal transmembrane domain (TM) region in gray. BH motifs are depicted using the same color code in the structure and in the motif architecture diagrams. Inset: Crystal structure of Bcl-xL in complex with ABT-737 (PDB code 2YXJ[95]). The figure was prepared with the program PyMOL.

Figure 2

Key Figure: Attributes and Interactome of the B Cell Lymphoma-2 (BCL-2) Homology 3 (BH3) Linear Motif Some or all BH3 motifs have the attributes of short linear motifs (SLiMs) associated with protein–protein binding events (left). However, BH3 is an unusual SLiM type as it is found in ordered proteins as well as intrinsically disordered proteins (IDPs) and achieves high-affinity binding to globular BCL-2-homologous proteins. BH3-mediated protein–protein interactions (PPIs) form an intricate network that controls the process of apoptotic cell death in metazoan organisms (right). Both pro- and antiapoptotic BCL-2 homologs have a globular domain structure, share common ancestry, and constitute binding sites (receptors) for BH3 motifs, which act as ligands to either activate the executioner proteins BAX/BAK or inhibit the antiapoptotic BCL-2 proteins. Both pro- and antiapoptotic BCL-2 homologs can receive a BH3 from BH3-only proteins and can also receive the BH3 from BAX/BAK (e.g., BAX–BAX or BAX–BCL-2 interactions). Redefining the BH3 cell death domain as a SLiM/molecular recognition feature (MoRF) leads to several testable predictions, including the possible existence of: (i) BH3-mediated protein–protein interactions in non-metazoan organisms (including prokaryotes); and (ii) non-BCL-2-like receptors for BH3 motifs. Because globular domains often bind to multiple linear motif classes, the question of whether BCL-2-homologous proteins can interact with other types of SLiMs, in addition to the BH3, is also open. The color scheme used to group the various attributes of the BH3 SLiM is as follows: grey, sequence features; green, binding features; pink, structural features; violet, other features.