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

• Published in: Nature communications Vol. 15; no. 1; pp. 10001 - 18
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 19-11-2024; Nature Publishing Group; Nature Portfolio
• Subjects: 49; 49/23; 631/1647/1888; 631/326/2522; 631/45/612/822; 631/61/338/469; Allosteric properties; Antimalarial agents; Binding; Biosensors; Design; Design factors; Flavonoids; Gene expression; Humanities and Social Sciences; Ligands; Methionine; multidisciplinary; Naltrexone; Naringenin; Quinine; Science; Science (multidisciplinary); Sensors; Transcription factors
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-54260-8

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.
The use of allosteric transcription factors (aTFs) as biosensors has been constrained by their limited natural ligand repertoire. Here, the authors report a method to screen large libraries of aTF variants to develop biosensors with altered specificities to non-native ligands.