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Conformational studies of the manganese transport regulator (MntR) from Bacillus subtilis using deuterium exchange mass spectrometry

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Abstract

The manganese transport regulator (MntR) of Bacillus subtilis is a metalloregulatory protein responsible for regulation of genes involved in manganese uptake by this organism. MntR belongs to the iron-responsive DtxR family, but is allosterically regulated by manganese and cadmium ions. Having previously characterized the metal binding affinities of this protein as well as the DNA-binding activation profiles for the relevant metal ions, we have focused the current study on investigating the structural changes of MntR in solution upon binding divalent transition metal ions. Deuterium exchange mass spectrometry was utilized to investigate the deuterium exchange dynamics between apo-MntR, Co2+-MntR, Cd2+-MntR, and Mn2+-MntR. Comparing the rates of deuteration of each metal-bound form of MntR reveals that the N-terminal DNA-binding motif is more mobile in solution than the C-terminal dimerization domain. Furthermore, significant protection from deuterium exchange is observed in the helices that contribute metal-chelating amino acids to form the metal binding site of MntR. In contrast, the bulk of the DNA-binding winged helix–turn–helix motif shows no difference in deuterium exchange upon metal binding. Mapping of the deuteration patterns onto the crystal structures of MntR yields insight into how metal binding affects the protein structure and complements earlier studies on the mechanism of MntR. Metal binding acts to rigidify MntR, thereby limiting the mobility of the protein and reducing the entropic cost of DNA binding.

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Abbreviations

CD:

Circular dichroism

DtxR:

Diphtheria toxin repressor

DXMS:

Deuterium-exchange mass spectrometry

GuHCl:

Guanidine hydrochloride

HTH:

Helix–turn–helix

MntR:

Manganese transport regulator

MS:

Mass spectrometry

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Acknowledgements

The authors thank Arthur Glasfeld (Reed College) for sharing crystallographic data and helpful discussions, John Helmann (Cornell University) for providing the MntR clone, and Patricia Jennings (University of California San Diego) for helpful discussions. This work was supported by the University of California, the University of California San Diego Academic Senate, and the Hellman Family Fund. S.M.C. is a Cottrell Scholar of the Research Corporation. NIH grants CA099835 and CA118595 are acknowledged (V.L.W.).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0358, USA

    Misha Golynskiy & Seth M. Cohen

  2. Department of Medicine and Biomedical Sciences, University of California, San Diego, La Jolla, CA, 92093-0358, USA

    Sheng Li & Virgil L. Woods Jr

Authors
  1. Misha Golynskiy
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  2. Sheng Li
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  3. Virgil L. Woods Jr
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  4. Seth M. Cohen
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Corresponding authors

Correspondence to Virgil L. Woods Jr or Seth M. Cohen.

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Golynskiy, M., Li, S., Woods, V.L. et al. Conformational studies of the manganese transport regulator (MntR) from Bacillus subtilis using deuterium exchange mass spectrometry. J Biol Inorg Chem 12, 699–709 (2007). https://doi.org/10.1007/s00775-007-0216-z

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  • DOI: https://doi.org/10.1007/s00775-007-0216-z

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