Highly multiplexed design of an allosteric transcription factor to sense new ligands
Abstract Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting new molecules because mutation of ligand-binding residues often disrupts...
Saved in:
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2024-11-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-54260-8 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849764085658288128 |
|---|---|
| author | Kyle K. Nishikawa Jackie Chen Justin F. Acheson Svetlana V. Harbaugh Phil Huss Max Frenkel Naia Novy Hailey R. Sieren Ella C. Lodewyk Daniel H. Lee Jorge L. Chávez Brian G. Fox Srivatsan Raman |
| author_facet | Kyle K. Nishikawa Jackie Chen Justin F. Acheson Svetlana V. Harbaugh Phil Huss Max Frenkel Naia Novy Hailey R. Sieren Ella C. Lodewyk Daniel H. Lee Jorge L. Chávez Brian G. Fox Srivatsan Raman |
| author_sort | Kyle K. Nishikawa |
| collection | DOAJ |
| description | Abstract Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting new molecules because mutation of ligand-binding residues often disrupts allostery. Here, we develop Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screen a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures – four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone – as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identifies biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design shows shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we develop cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid and scalable design of new biosensors, overcoming constraints of natural biosensors. |
| format | Article |
| id | doaj-art-e57ca63a79324d18b5975ab02734cb1b |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-e57ca63a79324d18b5975ab02734cb1b2025-08-20T03:05:14ZengNature PortfolioNature Communications2041-17232024-11-0115111810.1038/s41467-024-54260-8Highly multiplexed design of an allosteric transcription factor to sense new ligandsKyle K. Nishikawa0Jackie Chen1Justin F. Acheson2Svetlana V. Harbaugh3Phil Huss4Max Frenkel5Naia Novy6Hailey R. Sieren7Ella C. Lodewyk8Daniel H. Lee9Jorge L. Chávez10Brian G. Fox11Srivatsan Raman12Department of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-Madison711th Human Performance Wing, Air Force Research LaboratoryDepartment of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-Madison711th Human Performance Wing, Air Force Research LaboratoryDepartment of Biochemistry, University of Wisconsin-MadisonDepartment of Biochemistry, University of Wisconsin-MadisonAbstract Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting new molecules because mutation of ligand-binding residues often disrupts allostery. Here, we develop Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screen a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures – four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone – as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identifies biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design shows shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we develop cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid and scalable design of new biosensors, overcoming constraints of natural biosensors.https://doi.org/10.1038/s41467-024-54260-8 |
| spellingShingle | Kyle K. Nishikawa Jackie Chen Justin F. Acheson Svetlana V. Harbaugh Phil Huss Max Frenkel Naia Novy Hailey R. Sieren Ella C. Lodewyk Daniel H. Lee Jorge L. Chávez Brian G. Fox Srivatsan Raman Highly multiplexed design of an allosteric transcription factor to sense new ligands Nature Communications |
| title | Highly multiplexed design of an allosteric transcription factor to sense new ligands |
| title_full | Highly multiplexed design of an allosteric transcription factor to sense new ligands |
| title_fullStr | Highly multiplexed design of an allosteric transcription factor to sense new ligands |
| title_full_unstemmed | Highly multiplexed design of an allosteric transcription factor to sense new ligands |
| title_short | Highly multiplexed design of an allosteric transcription factor to sense new ligands |
| title_sort | highly multiplexed design of an allosteric transcription factor to sense new ligands |
| url | https://doi.org/10.1038/s41467-024-54260-8 |
| work_keys_str_mv | AT kyleknishikawa highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT jackiechen highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT justinfacheson highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT svetlanavharbaugh highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT philhuss highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT maxfrenkel highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT naianovy highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT haileyrsieren highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT ellaclodewyk highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT danielhlee highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT jorgelchavez highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT briangfox highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands AT srivatsanraman highlymultiplexeddesignofanallosterictranscriptionfactortosensenewligands |