bi300886q_si_001.pdf (311.76 kB)
Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR
journal contribution
posted on 2013-01-08, 00:00 authored by Khadine
A. Higgins, Heidi Q. Hu, Peter
T. Chivers, Michael J. MaroneyThe 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.