There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.
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Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII
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This work was supported by Biotechnology and Biological Sciences Research Council awards nos. BB/J017787/1, BB/R002118/1 and BB/L009226/1. Interactions with industrial partners were supported by Biotechnology and Biological Sciences Research Council (BBSRC) award no. BB/L013711/1 plus a financial contribution from Procter and Gamble (in association with an Industrial Partnership Award no. BB/J017787/1). K. Svedaite, Department of Biosciences, Durham University, provided technical assistance in the measurements of in vitro DNA affinities of ZntR and CueR. E. Pohl and C. Bain, both of Durham University Department of Chemistry, assisted with structure homology modeling and consideration of standard free-energy changes, respectively. Salmonella enterica serovar Typhimurium strain SL1344 was provided by J.S. Cavet, School of Biological Sciences, University of Manchester, Manchester, UK. E. Fioravanti, Mathematical Institute, University of Oxford, assisted with derivations shown in the Supplementary Note 2. All DNA sequencing was conducted by DBS genomics, Durham University.
J.C. and T.G.H. are employees of Procter and Gamble. The collaboration was supported by an Industrial Partnership Award from the BBSRC plus a financial contribution from Procter and Gamble (in association with BBSRC award no. BB/J017787/1).
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Supplementary Tables 1–5, Supplementary Figures 1–22
Excel Spreadsheet (with instructions) to enable calculation of fractional DNA occupancy
The Dynafit scripts
The supplementary equations and unique Supplementary Note 2 references
The MATLAB codes (with instructions), to determine the buffered metal concentration from given value(s) of θD or θDM
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Osman, D., Martini, M.A., Foster, A.W. et al. Bacterial sensors define intracellular free energies for correct enzyme metalation. Nat Chem Biol 15, 241–249 (2019). https://doi.org/10.1038/s41589-018-0211-4
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