Ni(II) and Co(II) sensing by Escherichia coli RcnR.
Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E. coli, to provide a better understanding how different nickel levels are sensed in E. coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.
Iwig
Jeffrey S
JS
Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics, Saint Louis, Missouri 63110, USA.
Leitch
Sharon
S
Herbst
Robert W
RW
Maroney
Michael J
MJ
Chivers
Peter T
PT
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009532:Nickel / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D002413:Cations, Divalent; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D003035:Cobalt; D009532:Nickel