Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L]
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 bindi...
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| Format: | Article |
| Language: | en_US |
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Elsevier
2012
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| Online Access: | http://hdl.handle.net/1721.1/73465 https://orcid.org/0000-0003-4074-8980 |
| _version_ | 1826194580963328000 |
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| author | Dutta, Sanjib Gulla, Stefano Fire, Emiko Keating, Amy E. Chen, T. Scott Grant, Robert A |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Dutta, Sanjib Gulla, Stefano Fire, Emiko Keating, Amy E. Chen, T. Scott Grant, Robert A |
| author_sort | Dutta, Sanjib |
| collection | MIT |
| description | 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 α-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[subscript 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[subscript 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[subscript L], Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x[subscript 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[subscript L] binders. |
| first_indexed | 2024-09-23T09:58:28Z |
| format | Article |
| id | mit-1721.1/73465 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:58:28Z |
| publishDate | 2012 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/734652022-09-26T14:54:55Z Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L] Dutta, Sanjib Gulla, Stefano Fire, Emiko Keating, Amy E. Chen, T. Scott Grant, Robert A Massachusetts Institute of Technology. Department of Biology Dutta, Sanjib Gulla, Stefano Chen, Tsan Chou Scott Fire, Emiko Grant, Robert A. Keating, Amy E. 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 α-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[subscript 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[subscript 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[subscript L], Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x[subscript 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[subscript L] binders. National Institute of General Medical Sciences (U.S.) (Award GM084181) National Institute of General Medical Sciences (U.S.) (Award P50-GM68762) National Center for Research Resources (U.S.) (Award RR-15301) 2012-09-28T14:10:16Z 2012-09-28T14:10:16Z 2010-04 2010-03 Article http://purl.org/eprint/type/JournalArticle 0022-2836 1089-8638 http://hdl.handle.net/1721.1/73465 Dutta, Sanjib et al. “Determinants of BH3 Binding Specificity for Mcl-1 Versus Bcl-x[subscript L].” Journal of Molecular Biology 398.5 (2010): 747–762. https://orcid.org/0000-0003-4074-8980 en_US http://dx.doi.org/10.1016/j.jmb.2010.03.058 Journal of Molecular Biology Creative Commons Attribution-Noncommercial-Share Alike 3.0 http://creativecommons.org/licenses/by-nc-sa/3.0/ application/pdf Elsevier PMC |
| spellingShingle | Dutta, Sanjib Gulla, Stefano Fire, Emiko Keating, Amy E. Chen, T. Scott Grant, Robert A Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L] |
| title | Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L] |
| title_full | Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L] |
| title_fullStr | Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L] |
| title_full_unstemmed | Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L] |
| title_short | Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x[subscript L] |
| title_sort | determinants of bh3 binding specificity for mcl 1 versus bci x subscript l |
| url | http://hdl.handle.net/1721.1/73465 https://orcid.org/0000-0003-4074-8980 |
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