Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-xL

Abstract

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-x L vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x L selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bclx L , Bcl-2, Bcl-w and Bfl-1, whereas Bcl-x L selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-x L binders.

BH3 Mimetics: Status of the Field and New Developments

“…Dutta and colleagues compared the sequence determinants of BH3-binding specificity for MCL-1 versus BCL-X L (by screening of BH3 peptide libraries) and identified BH3 peptides that selectively interact with either MCL-1 or BCL-X L (or both) with high affinity. By examining the effects of 170 point mutations in the BH3 region of BIM, these investigators characterized important sequence features and constructed a predictive model that explains much of the binding specificity (56). Together, these findings may be used to improve the binding affinity of either MCL-1-specific or pan-BH3 mimetics.…”

Molecular docking studies of anti-apoptotic BCL-2, BCL-XL, and MCL-1 proteins with ginsenosides from Panax ginseng

“…For docking studies three-dimensional structures of antiapoptotic proteins such as BCL-2, BCL-XL, and MCL-1 were retrieved from the RCSB protein data bank 13,21 with their respective PDB id's 2W3L 22 , 2YXJ 23 , and 3KZ0 24 . These protein structures were determined using X-ray diffraction methods.…”

The Functional Differences between Pro-survival and Pro-apoptotic B Cell Lymphoma 2 (Bcl-2) Proteins Depend on Structural Differences in Their Bcl-2 Homology 3 (BH3) Domains

“…Subtle differences in some of the conserved residues (i.e. different hydrophobic residues at h1, h3, or h4) as well as in the intervening residues contribute to the significant differences in selectivity in interactions between BH3 domains and pro-survival proteins (15,(25)(26)(27)(28)(29). Indeed, some BH3 sequences are promiscuous and engage all pro-survival proteins with high (low nanomolar) affinity, whereas others are more selective, only engaging subsets of pro-survival proteins avidly (30 -32).…”