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. 2023 Jun 8;19(9):1147–1157. doi: 10.1038/s41589-023-01350-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a. Zoom-in on the NAD⁺ binding site of the X-ray structure of LigA from Enterococcus faecalis (efLigA) bound to NAD⁺ (PDB ID: 1TAE). The structure is represented as cartoon (light blue) and NAD⁺ (green) and residues involved in the binding of NAD⁺ (Y222 and V284, light blue) are represented as sticks. *Y222 and V284 of efLigA correspond to Y226 and V292 of LigA from Thermus thermophilus (ttLigA). b. NAD⁺ titrations of sensor variants labeled with TMR. ttLigA^D carries the extra mutations K117L and D289N rendering it catalytically inactive. Mutations Y226W and Y292A shift the sensor response towards the range of free intracellular NAD⁺. c. FRET ratios of NAD⁺ sensors differing in the number of interface mutations (Supplementary Table S5) in presence (+NAD⁺) or absence (-NAD⁺) of 1 mM NAD⁺. d. Maximal FRET/eGFP ratio change (^MaxΔR/R₀) of NAD+ sensors differing in the number of interface mutations. Asterisk indicates construct corresponding to the final sensor ChemoG-NAD. e. Titration of ChemoG-NAD with NAD⁺ or structurally related molecules. f. Titration of ChemoG-NAD_SiR with NAD+ in presence of different structurally related molecules. g. Titration of ChemoG-NAD_SiR with NAD⁺ in presence of 1 mM AMP, ADP or ATP. h, i. NAD⁺ titrations of ChemoG-NAD_SiR at different temperatures (h) or at different pH (i). For all graphs, the means of 3 technical replicates ±s.d. are shown.

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