Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L]
Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Redesigned proteins could be used as resear...
| Main Authors: | , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Elsevier
2016
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| Online Access: | http://hdl.handle.net/1721.1/101270 https://orcid.org/0000-0003-4074-8980 |
| _version_ | 1811006969878675456 |
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| author | Chen, T. Scott Palacios, Hector Keating, Amy E. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Chen, T. Scott Palacios, Hector Keating, Amy E. |
| author_sort | Chen, T. Scott |
| collection | MIT |
| description | Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Redesigned proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to reengineer the multi-specific anti-apoptotic protein Bcl-x[subscript L] to remove its interactions with many of its binding partners, making it a high-affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-x[subscript L] sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-x[subscript L] variants, we successfully identified a protein with ~ 1000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best redesigned protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems. |
| first_indexed | 2024-09-23T17:00:33Z |
| format | Article |
| id | mit-1721.1/101270 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T17:00:33Z |
| publishDate | 2016 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1012702022-10-03T09:47:29Z Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L] Chen, T. Scott Palacios, Hector Keating, Amy E. Massachusetts Institute of Technology. Department of Biology Chen, T. Scott Palacios, Hector Keating, Amy E. Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Redesigned proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to reengineer the multi-specific anti-apoptotic protein Bcl-x[subscript L] to remove its interactions with many of its binding partners, making it a high-affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-x[subscript L] sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-x[subscript L] variants, we successfully identified a protein with ~ 1000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best redesigned protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems. National Institutes of Health (U.S.) (Award GM084181) 2016-02-25T03:18:14Z 2016-02-25T03:18:14Z 2012-11 2012-11 Article http://purl.org/eprint/type/JournalArticle 00222836 1089-8638 http://hdl.handle.net/1721.1/101270 Chen, T. Scott, Hector Palacios, and Amy E. Keating. “Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L].” Journal of Molecular Biology 425, no. 1 (January 2013): 171–185. https://orcid.org/0000-0003-4074-8980 en_US http://dx.doi.org/10.1016/j.jmb.2012.11.009 Journal of Molecular Biology Creative Commons Attribution-Noncommercial-NoDerivatives http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier PMC |
| spellingShingle | Chen, T. Scott Palacios, Hector Keating, Amy E. Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L] |
| title | Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L] |
| title_full | Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L] |
| title_fullStr | Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L] |
| title_full_unstemmed | Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L] |
| title_short | Structure-Based Redesign of the Binding Specificity of Anti-Apoptotic Bcl-x[subscript L] |
| title_sort | structure based redesign of the binding specificity of anti apoptotic bcl x subscript l |
| url | http://hdl.handle.net/1721.1/101270 https://orcid.org/0000-0003-4074-8980 |
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