Role of the N-terminus in determining metal-specific responses in the E. coli Ni- and Co-responsive metalloregulator, RcnR

Abstract

RcnR (resistance to cobalt and nickel regulator) is a 40-kDa homotetrameric protein and metalloregulator that controls the transcription of the Co(II) and Ni(II) exporter, RcnAB, by binding to DNA as an apoprotein and releasing DNA in response to specifically binding Co(II) and Ni(II) ions. Using X-ray absorption spectroscopy (XAS) to examine the structure of metals bound and lacZ reporter assays of the transcription of RcnA in response to metal binding, in WT and mutant proteins, the roles of coordination number, ligand selection, and residues in the N-terminus of the protein were examined as determinants in metal ion recognition. The studies show that the cognate metal ions, Co(II) and Ni(II), which bind in (N/O)(5)S six-coordinate sites, are distinguished from non-cognate metal ions (Cu(I) and Zn(II)), which bind only three protein ligands and one anion from the buffer, by coordination number and ligand selection. Using mutations of residues near the N-terminus, the N-terminal amine is shown to be a ligand of the cognate metal ions that is missing in the complexes with non-cognate metal ions. The side chain of His3 is also shown to play an important role in distinguishing metal ions. The imidazole group is shown to be a ligand in the Co(II) RcnR complex, but not in the Zn(II) complex. Further, His3 does not appear to bind to Ni(II), providing a structural basis for the differential regulation of RcnAB by the two cognate ions. The Zn(II) complexes change coordination number in response to the residue in position three. In H3C-RcnR, the Zn(II) complex is five-coordinate, and in H3E-RcnR the Zn(II) ion is bound to six protein ligands. The metric parameters of this unusual Zn(II) structure resemble those of the WT-Ni(II) complex, and the mutant protein is able to regulate expression of RcnAB in response to binding the non-cognate ion. The results are discussed within a protein allosteric model for gene regulation by metalloregulators.

Figure 1

The effects of H3C- and H3E-RcnR mutant proteins on Co(II) and Ni(II) responsiveness.

Figure 2

LacZ reporter assay showing the expression of P_rcnA with increased Zn(II) ion concentrations in the H3E mutant protein compared to the wild-type protein.

Figure 3

XANES overlay of metal complexes of RcnR proteins in buffer with 20 mM Hepes, 300 mM NaBr and 10% glycerol (except where noted): WT-RcnR (red), A2* (blue), H3L (green), H3L-OAc (cyan), H3C (purple) and H3E (orange).

Figure 4

Metal complexes of wild-type RcnR in buffer with 20 mM Hepes, 300 mM NaCl, and 10 % glycerol at pH 7.0. Left: Fourier-filtered XAS data (colored lines) and best fits (black lines) from Table 2. Right: Unfiltered k³-weighted EXAFS spectra and fits.

Figure 5

Metal complexes of wild-type RcnR in buffer with 20 mM Hepes, 300 mM NaBr, and 10% glycerol at pH 7.0. Left: Fourier filtered XAS data (colored lines) and best fits (black lines) from Table 2. Right: Unfiltered k³-weighted EXAFS spectra and fits.

Figure 6

Metal complexes of A2*-RcnR in buffer with 20 mM Hepes, 300 mM NaBr, and 10% glycerol at pH 7.0. Left: Fourier filtered XAS data (colored lines) and best fits (black lines) from Table 3. Right: Unfiltered k³-weighted EXAFS spectra and fits.

Figure 7

Metal complexes of H3L-RcnR in buffer with 20mM Hepes, 300mM NaBr /¹NaOAc, and 10% glycerol at pH 7.0. Left: Fourier filtered XAS data (colored lines) and best fits (black lines) from Table 3. Right: Unfiltered k³ weighted EXAFS spectra and fits.

Figure 8

Metal complexes of H3C-RcnR in buffer with 20mM Hepes, 300mM NaBr, and 10% glycerol at pH 7.0. Left: Fourier filtered XAS data (colored lines) and best fits (black lines) from Table 2. Right: Unfiltered k³-weighted EXAFS spectra and fits.

Figure 9

Metal complexes of H3E-RcnR in buffer with 20mM Hepes, 300mM NaBr, and 10% glycerol at pH 7.0. Left: Fourier filtered XAS data (colored lines) and best fits (black lines) from Table 3. Right: Unfiltered k³-weighted EXAFS spectra and fits.