Genome-Wide Prediction and Validation of Peptides That Bind Human Prosurvival Bcl-2 Proteins

Abstract

Programmed cell death is regulated by interactions between pro-apoptotic and prosurvival members of the Bcl-2 family. Pro-apoptotic family members contain a weakly conserved BH3 motif that can adopt an alpha-helical structure and bind to a groove on prosurvival partners Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. Peptides corresponding to roughly 13 reported BH3 motifs have been verified to bind in this manner. Due to their short lengths and low sequence conservation, BH3 motifs are not detected using standard sequence-based bioinformatics approaches. Thus, it is possible that many additional proteins harbor BH3-like sequences that can mediate interactions with the Bcl-2 family. In this work, we used structure-based and data-based Bcl-2 interaction models to find new BH3-like peptides in the human proteome. We used peptide SPOT arrays to test candidate peptides for interaction with one or more of the prosurvival proteins Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. For the 36 most promising array candidates, we quantified binding to all five human receptors using direct and competition binding assays in solution. All 36 peptides showed evidence of interaction with at least one prosurvival protein, and 22 peptides bound at least one prosurvival protein with a dissociation constant between 1 and 500 nM; many peptides had specificity profiles not previously observed. We also screened the full-length parent proteins of a subset of array-tested peptides for binding to Bcl-xL and Mcl-1. Finally, we used the peptide binding data, in conjunction with previously reported interactions, to assess the affinity and specificity prediction performance of different models.

Locating Herpesvirus Bcl-2 Homologs in the Specificity Landscape of Anti-Apoptotic Bcl-2 Proteins

“…22,30,32 Recently, DeBartolo et al reported dissociation constants for the five human Bcl-2 homologs binding to 36 new, computationally identified candidate BH3 peptides from the human proteome. 46 In Fig. 3b, we compare the binding patterns of the three viral Bcl-2 homologs to those of the human homologs for these 36 BH3-like peptides.…”

“…Specifically, we designed the libraries using mutational data from SPOT arrays and predictions made using STATIUM, a statistical potential that can evaluate mutations based on analysis of human or viral Bcl-2 crystal structures or homology models. 46,48 We also used Illumina sequencing data from a yeast surface display library of Bim BH3 variants previously screened for KSBcl-2 binding. 25 The computationally assisted library design process (described in further detail in the Materials and Methods) focused on including residues that were tolerated by the viral Bcl-2 proteins and that weakened binding to Mcl-1 and, for the BHRF1 library, Bfl-1.…”

“…Proteome-derived BH3 peptides (26-mers, N-fluoresceinated, C-amidated) in Fig. 3 were the same as those used by DeBartolo et al 46 Direct fluorescence anisotropy experiments were performed as in Foight et al, with a titration of twelve receptor concentrations, with a maximum concentration of 1 μM receptor for the library peptide curves and 3 μM for the proteome peptide curves. 25 C-myc-tagged receptors were used for all Bcl-2 homologs, with the exception of M11, for which a BAP and His 6 -tagged construct was used.…”

Template-based prediction of protein function

“…Most notably, template-based methods are having considerable biological impact, and have been applied to identify, almost always with experimental validation, previously unknown protein-protein interactions [13*,40*], RNA-binding proteins [23*], membrane binding-proteins [48*], enzyme families [49*], drug targets [21*], to analyze biological pathways [50,51] and to understand the relationship between genetic variation and disease [52*]. Protein structure is, of course, a key component of these approaches.…”

“…identifying residues that occur more frequently in certain types of interfaces, as compared to a non-interfacial background. This approach has recently been used to identify novel proteins that interact with DNA or RNA [22,23*] and novel interactors of Bcl2 proteins [40*]. In another approach, a large set of protein-peptide structures was examined to construct, for each of the twenty amino acids, a three-dimensional grid of boxes containing the set of chemical groups that more frequently interact with that amino acid in protein-peptide interfaces [41].…”