Structural elements of metal selectivity in metal sensor proteins

Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR are homologous zinc/cobalt-responsive and nickel/cobalt-responsive transcriptional repressors in vivo, respectively, and members of the ArsR/SmtB superfamily of prokaryotic metal sensor proteins. We show here that Zn(II) is the most potent negative allosteric regulator of czr operator/promoter binding in vitro with the trend Zn(II)>Co(II)much greater thanNi(II), whereas the opposite holds for the binding of NmtR to the nmt operator/promoter, Ni(II)>Co(II)>Zn(II). Characterization of the metal coordination complexes of CzrA and NmtR by UV/visible and x-ray absorption spectroscopies reveals that metals that form four-coordinate tetrahedral complexes with CzrA [Zn(II) and Co(11)] are potent regulators of DNA binding, whereas metals that form five- or six-coordinate complexes with NmtR [Ni(II) and Co(II)] are the strongest allosteric regulators in this system. Strikingly, the Zn(II) coordination complexes of CzrA and NmtR cannot be distinguished from one another by x-ray absorption spectroscopy, with the best fit a His-3-carboxylate complex in both cases. Inspection of the primary structures of CzrA and NmtR, coupled with previous functional data, suggests that three conserved His and one Asp from the C-terminal alpha5 helix donate ligands to create a four-coordinate complex in both CzrA and NmtR, with NmtR uniquely capable of expanding its coordination number in the Ni(II) and Co(II) complexes by recruiting additional His ligands from a C-terminal extension of the alpha5 helix.