Highly multiplexed design of an allosteric transcription factor to sense new ligands

Nishikawa, Kyle K.; Chen, Jackie; Acheson, Justin F.; Harbaugh, Svetlana V.; Huss, Phil; Frenkel, Max; Novy, Nathan; Sieren, Hailey R.; Lodewyk, Ella C.; Lee, Daniel H.; Chávez, Jorge L.; Fox, Brian G.; Raman, Srivatsan

doi:10.1038/s41467-024-54260-8

Title: Highly multiplexed design of an allosteric transcription factor to sense new ligands

Journal Article · 18 November 2024 · Nature Communications

DOI: https://doi.org/10.1038/s41467-024-54260-8 · OSTI ID:2506971

Nishikawa, Kyle K. ^[1]; Chen, Jackie ^[1];

^[1]; Harbaugh, Svetlana V. ^[2]; Huss, Phil ^[1]; Frenkel, Max ^[1]; Novy, Nathan ^[1]; Sieren, Hailey R. ^[3]; Lodewyk, Ella C. ^[3]; Lee, Daniel H. ^[3]; Chávez, Jorge L. ^[2]; Fox, Brian G. ^[1];

^[1]

University of Wisconsin-Madison, WI (United States)
Air Force Research Laboratory, Wright Patterson Air Force Base, OH (United States)
University of Wisconsin-Madison, WI (United States); State of Wisconsin Department of Workforce Development, Madison, WI (United States)

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.

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Research Organization:: Great Lakes Bioenergy Research Center (GLBRC), Madison, WI (United States)

Sponsoring Organization:: National Institute of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER); United States Army Research Office (ARO)

Grant/Contract Number:: AC02-06CH11357; SC0018409

OSTI ID:: 2506971

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 15; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Title: Highly multiplexed design of an allosteric transcription factor to sense new ligands

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