Mapping the BH3 Binding Interface of Bcl-x_L, Bcl-2, and Mcl-1 Using Split-Luciferase Reassembly

Abstract

The recognition of helical BH3 domains by Bcl-2 homology (BH) receptors plays a central role in apoptosis. The residues that determine specificity or promiscuity in this interactome are difficult to predict from structural and computational data. Using a cell free split-luciferase system, we have generated a 276 pairwise interaction map for 12 alanine mutations at the binding interface for three receptors, Bcl-xL, Bcl-2, and Mcl-1, and interrogated them against BH3 helices derived from Bad, Bak, Bid, Bik, Bim, Bmf, Hrk, and Puma. This panel, in conjunction with previous structural and functional studies, starts to provide a more comprehensive portrait of this interactome, explains promiscuity, and uncovers surprising details; for example, the Bcl-xL R139A mutation disrupts binding to all helices but the Bad-BH3 peptide, and Mcl-1 binding is particularly perturbed by only four mutations of the 12 tested (V220A, N260A, R263A, and F319A), while Bcl-xL and Bcl-2 have a more diverse set of important residues depending on the bound helix.

Docking-based identification of small-molecule binding sites at protein-protein interfaces

“…For some of the predicted hot-spot residues, there is experimental evidence in the literature. For instance, Bcl-xL F146 residue is predicted here as a hot-spot, which is confirmed in previous experimental studies [84] , [85] , [86] . Several hot-spots have been experimentally identified for E2 (Y19, Q24, E39, Y99 and E100) [87] , and we successfully predict two of them (Y19 and E100).…”

Computational Modeling as a Tool to Investigate PPI: From Drug Design to Tissue Engineering

“…Bad and Bmf preferently bind Bcl-2, Bcl-xL and Bcl-w, whereas Noxa bind only Mcl-1 and Bfl1/A1 and Bid, Bik and Hrk bind Bcl-xL, Bcl-1 and Bfl1/A1 ( Chen et al, 2005 ). This differential profile is due to differences in their sequence, since mutations can reverse binding preferences ( Campbell et al, 2015 ). Numerous structural and computational studies have been performed to understand the nature of the binding preferences between BH3 peptides the diverse members of the Bcl-2 family, providing a deeper insight into the nature of these interactions key to design BH3 peptide surrogates and peptidomimetics ( Lama and Sankararamakrishnan, 2011 ; Ivanov et al, 2016 ; Zhang et al, 2018 ; Vila-Julià et al, 2020 ).…”

Bcl-xL Dynamics under the Lens of Protein Structure Networks

“…Of particular interest are F146, experimentally observed as important for the binding of the BH3-only proteins to Bcl-x L , , and C151, whose mutation C151Y features a high pathogenicity score according to the REVEL ensemble predictor (Figure C).…”

“…In particular, they reported Q125 as a key residue sensitive to the allosteric effect induced by p53 binding, enforcing the notion that the interaction with p53 influences the binding mechanism of PUMA. Moreover, the work of Campbell and coworkers with a cell-free split-luciferase assay identified three of the PUMA-induced hubs (E129, L130, R139) as crucial residues for the binding of several BH3-only proteins, including PUMA. All of them are identified as hubs only in the ensembles of the Bcl-x L –PUMA complex, reinforcing the hypothesis of a prominent role in mediating the binding process.…”