Effects of select histidine to cysteine mutations on transcriptional regulation by Escherichia coli RcnR

Abstract

The RcnR metalloregulator represses the transcription of the Co(II) and Ni(II) exporter, RcnAB. Previous studies have shown that Co(II) and Ni(II) bind to RcnR in six-coordinate sites, resulting in derepression. Here, the roles of His60, His64, and His67 in specific metal recognition are examined. His60 and His64 correspond to ligands that are important for Cu(I) binding in the homologous Cu(I)-responsive metalloregulator, CsoR. These residues are known to be functionally important in RcnR transcriptional regulation. X-ray absorption spectroscopy (XAS) was used to examine the structure of bound cognate and noncognate metal ions, and lacZ reporter assays were used to assess the transcription of rcnA in response to metal binding in the three His → Cys mutations, H60C, H64C, and H67C. These studies confirm that both Ni(II) and Co(II) use His64 as a ligand. H64C-RcnR is also the only known mutant that retains a Co(II) response while eliminating the response to Ni(II) binding. XAS data indicate that His60 and His67 are potential Co(II) ligands. The effects of the mutations of His60, His64, and His67 on the structures of the noncognate metal ions [Zn(II) and Cu(I)] reveal that these residues have distinctive roles in binding noncognate metals. None of the His → Cys mutants in RcnR confer any response to Cu(I) binding, including H64C-RcnR, where the ligands involved in Cu(I) binding in CsoR are present. These data indicate that while the secondary, tertiary, and quaternary structures of CsoR and RcnR are quite similar, small changes in primary sequence reveal that the specific mechanisms involved in metal recognition are quite different.

Figure 1

Sequence alignment of E. coli RcnR, M. tuberculosis CsoR and Synechocystis PCC 6803 InrS generated using ClustalW (27). The metal binding residues of RcnR and CsoR are highlighted as well as the corresponding residues in InrS. Non conserved residues in RcnR are boxed.

Figure 2

LacZ reporter assay showing the effect of the H60C-, H64C- and H67C-RcnR mutations on the expression of P_rcnA in response to binding metal ions.

Figure 3

XANES spectra of metal complexes of RcnR proteins in buffer with 20 mM Hepes, 300 mM NaBr and 10% glycerol: H60C (red), H64C (dark green), H67C (purple) and WT from ref. (black). XANES spectra in buffer replacing NaBr with 300 mM NaOAc: H60C (orange), H64C (aqua), H67C (light green).

Figure 4

K-edge XAS spectra of H60C RcnR metal complexes in buffer containing 20 mM Hepes, 300 mM NaBr/¹NaOAc and 10% glycerol at pH 7.0. For Cu(I), the buffer also contained 2 mM TCEP. Left: Fourier transformed EXAFS data (colored lines) and fits (black lines). Right: Unfiltered k³-weighted EXAFS spectra and fits.

Figure 5

K-edge XAS spectra of H64C-RcnR metal complexes in buffer containing 20 mM Hepes, 300 mM NaBr/¹NaOAc and 10% glycerol at pH 7.0. For Cu(I), the buffer also contained 2 mM TCEP. Left: Fourier transformed EXAFS data (colored lines) and fits (black lines). Right: Unfiltered k³-weighted EXAFS spectra and fits.

Figure 6

K-edge XAS spectra of H67C RcnR metal complexes in buffer containing 20 mM Hepes, 300 mM NaBr/¹NaOAc and 10% glycerol at pH 7.0. For Cu(I), the buffer also contained 2 mM TCEP. Left: Fourier-transformed EXAFS data (colored lines) and fits (black lines). Right: Unfiltered k³-weighted EXAFS spectra and fits.