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Title: Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L]

Abstract

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-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 proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival 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{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub 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 boundmore » to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub 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 versus Bcl-x{sub L} binders.« less

Authors:
; ; ; ; ;  [1]
  1. MIT
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1002470
Resource Type:
Journal Article
Journal Name:
J. Mol. Biol.
Additional Journal Information:
Journal Volume: 398; Journal Issue: (5) ; 05, 2010; Journal ID: ISSN 0022-2836
Country of Publication:
United States
Language:
ENGLISH
Subject:
36 MATERIALS SCIENCE; AFFINITY; APOPTOSIS; BINDERS; CRYSTAL STRUCTURE; DEATH; DESIGN; FORECASTING; GENE MUTATIONS; PEPTIDES; PROTEINS; SCREENS; SPECIFICITY; YEASTS

Citation Formats

Dutta, Sanjib, Gullá, Stefano, Chen, T Scott, Fire, Emiko, Grant, Robert A, and Keating, Amy E. Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L]. United States: N. p., 2010. Web. doi:10.1016/j.jmb.2010.03.058.
Dutta, Sanjib, Gullá, Stefano, Chen, T Scott, Fire, Emiko, Grant, Robert A, & Keating, Amy E. Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L]. United States. https://doi.org/10.1016/j.jmb.2010.03.058
Dutta, Sanjib, Gullá, Stefano, Chen, T Scott, Fire, Emiko, Grant, Robert A, and Keating, Amy E. 2010. "Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L]". United States. https://doi.org/10.1016/j.jmb.2010.03.058.
@article{osti_1002470,
title = {Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L]},
author = {Dutta, Sanjib and Gullá, Stefano and Chen, T Scott and Fire, Emiko and Grant, Robert A and Keating, Amy E},
abstractNote = {Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-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 proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival 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{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub 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 Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub 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 versus Bcl-x{sub L} binders.},
doi = {10.1016/j.jmb.2010.03.058},
url = {https://www.osti.gov/biblio/1002470}, journal = {J. Mol. Biol.},
issn = {0022-2836},
number = (5) ; 05, 2010,
volume = 398,
place = {United States},
year = {Fri Jun 25 00:00:00 EDT 2010},
month = {Fri Jun 25 00:00:00 EDT 2010}
}