Ni(II) Sensing by RcnR Does Not Require an FrmR-Like Intersubunit Linkage

Area of Science:

• Biochemistry
• Molecular Biology
• Metalloprotein regulation

Background:

• E. coli RcnR is a DNA binding protein regulating metal exporter genes.
• RcnR binds cognate metal ions Ni(II) and Co(II) at subunit interfaces.
• Previous models proposed metal-mediated cross-linking is crucial for RcnR's allosteric response.

Purpose of the Study:

• To investigate the role of the interfacial metal binding site and Ni(II) coordination in RcnR's allosteric regulation.
• To determine if the molecular mechanisms of RcnR regulation by Ni(II) and Co(II) are distinct.

Main Methods:

• Site-directed mutagenesis to disrupt subunit cross-linking.
• X-ray absorption spectroscopy (XAS) for structural analysis.
• LacZ reporter assays for gene expression studies.
• Isothermal titration calorimetry (ITC) for metal binding analysis.

Main Results:

• Disrupting the Ni(II) cross-link did not affect RcnR's allosteric response to Ni(II).
• The interfacial binding site is crucial for Co(II)'s allosteric response, but not Ni(II)'s.
• His33 is not a direct ligand for Ni(II), Co(II), or Zn(II) in wild-type RcnR.
• Tight metal binding was maintained in the mutated variants.

Conclusions:

• The allosteric mechanisms of RcnR differ for Ni(II) and Co(II).
• Interfacial metal binding and cross-linking are not universally essential for RcnR function.
• This study refines our understanding of metallosensor mechanisms.