Mycobacterium tuberculosis is an obligate human respiratory pathogen that encodes approximately 10 arsenic repressor (ArsR) family regulatory proteins that allow the organism to respond to a wide range of changes in its immediate microenvironment. How individual ArsR repressors have evolved to respond to selective stimuli is of intrinsic interest. The Ni(II)/Co(II)-specific repressor NmtR and related actinomycete nickel sensors harbor a conserved N-terminal α-NH 2-Gly2-His3-Gly4 sequence. Here, we present the solution structure of homodimeric apo-NmtR and show that the core of the molecule adopts a typical winged-helix ArsR repressor (α1-α2-α3-αR- β1-β2-α5) "open conformation" that is similar to that of the related zinc sensor Staphylococcus aureus CzrA, but harboring long, flexible N-terminal (residues 2-16) and C-terminal (residues 109-120) extensions. Binding of Ni(II) to the regulatory sites induces strong paramagnetic broadening of the α5 helical region and the extreme N-terminal tail to residue 10. Ratiometric pulse chase amidination mass spectrometry reveals that the rate of amidination of the α-amino group of Gly2 is strongly attenuated in the Ni(II) complex relative to the apo state and noncognate Zn(II) complex. Ni(II) binding also induces dynamic disorder on the microsecond to millisecond time scale of key DNA interacting regions that likely contributes to the negative regulation of DNA binding by Ni(II). Molecular dynamics simulations and quantum chemical calculations reveal that NmtR readily accommodates a distal Ni(II) hexacoordination model involving the α-amine and His3 of the N-terminal region and α5 residues Asp91′, His93′, His104, and His107, which collectively define a new metal sensing site configuration in ArsR family regulators.
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