Solution Structure of Human BCL-w

Abstract

The structure of human BCL-w, an anti-apoptotic member of the BCL-2 family, was determined by tripleresonance NMR spectroscopy and molecular modeling. Introduction of a single amino acid substitution (P117V) significantly improved the quality of the NMR spectra obtained. The cytosolic domain of BCL-w consists of 8 ␣-helices, which adopt a fold similar to that of BCL-x L , BCL-2, and BAX proteins. Pairwise root meant square deviation values were less than 3 Å for backbone atoms of structurally equivalent regions. Interestingly, the Cterminal helix ␣8 of BCL-w folds into the BH3-binding hydrophobic cleft of the protein, in a fashion similar to the C-terminal transmembrane helix of BAX. A peptide corresponding to the BH3 region of the pro-apoptotic protein, BID, could displace helix ␣8 from the BCL-w cleft, resulting in helix unfolding. Deletion of helix ␣8 increased binding affinities of BCL-w for BAK and BID BH3-peptides, indicating that this helix competes for peptide binding to the hydrophobic cleft. These results suggest that although the cytosolic domain of BCL-w exhibits an overall structure similar to that of BCL-x L and BCL-2, the unique organization of its C-terminal helix may modulate BCL-w interactions with pro-apoptotic binding partners.Programmed cell death or apoptosis is an essential and integral part of development and tissue turnover under normal physiological conditions and perturbations to this process result in various diseases ranging from autoimmune disorders to cancer (1-4). Apoptosis is generally regulated by the fine balance and interplay among members of the BCL-2 family of proteins in a cell and tissue specific manner (5, 6). This family comprises nearly 20 proteins, which share one or more short regions of sequence homology, the BCL-2 homology regions (BH1, BH2, BH3 and BH4) that are essential for their function (5). The family is further divided into three subfamilies: antiapoptotic members including BCL-2, BCL-x L , BCL-w, MCL-1, and A1, which contain all four BH regions; pro-apoptotic members that contain BH1, BH2 and BH3 regions and function as death effectors (BAX and BAK); and pro-apoptotic BH3-only members (e.g. BAD, BID, BIM, BIK), which couple upstream death stimuli, such as tumor necrosis factor family receptor stimultation or p53-mediated stress signals, to regulation of the multi-BH region members (7). The ratio of anti-apoptotic versus pro-apoptotic members often determines cell death or survival in response to these signals, and alterations in these ratios can result in disease susceptibility. Enhanced expression of anti-apoptotic members occurs in many cancers. For example, up-regulation of BCL-2 in follicular lymphoma, leukemia, breast, and lung cancers (8 -11), BCL-x L in liver cancer, myeloid and T-cell leukemias (9), MCL-1 in multiple myeloma (12) and ovarian carcinoma (13), and BCL-w in colorectal carcinomas (14) has been observed and likely contributes to both the etiology of these cancers and the development of chemoresistance, which is frequently encountered.To da...

Solution Structure of Prosurvival Mcl-1 and Characterization of Its Binding by Proapoptotic BH3-only Ligands

“…The equivalent hydrogen bond in Mcl-1 would be disrupted, as the corresponding residues are Lys-215 and His-233, respectively, and this may contribute to the open conformation found in Mcl-1⌬NC23. Like Mcl-1, Bcl-w also has ␣3 and ␣4 in an open conformation, but here the C-terminal residues occupy the binding groove and may influence the conformation (23,38). These differences in the binding groove likely contribute to the specific binding properties of Mcl-1.…”

Comparison of chemical inhibitors of antiapoptotic Bcl-2-family proteins

“…Previous structural studies of Bcl-2, Bcl-X L , Bcl-W, and Mcl-1 have been performed using proteins in which the C-terminal membrane-insertion domain was similarly omitted from the recombinant protein. [7][8][9] Proteins were purified by one-step affinity chromatography using glutathione-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with Coomassie staining demonstrated that the resulting proteins were 485% intact (Figure 1a).…”

Bcl-2 Homodimerization Involves Two Distinct Binding Surfaces, a Topographic Arrangement That Provides an Effective Mechanism for Bcl-2 to Capture Activated Bax

“…Three-dimensional structures of monomeric antiapoptotic proteins such as Bcl-x L , Bcl-2, Bcl-w, and CED-9 as well as proapoptotic proteins such as Bax and Bid have been determined (5)(6)(7)(8)(9)(10)(11)(12). A hydrophobic surface groove common to the structures of all of the antiapoptotic proteins is encircled by five helices (helices 2-5 and 8) and connecting loops.…”