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 de-repression. 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. XAS was used to examine the structure of bound cognate and non-cognate 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 mutation 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 residues on the structures of the non-cognate metal ions (Zn(II) and Cu(I)) reveals that these residues have distinctive roles in binding non-cognate 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.

Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)

“…The same is more generally true for Ni(II)/Co(II)-specific RcnR (group IIa; Fig. 1) versus M. tuberculosis CsoR (48). Consistent with this, our functional analysis of R65A and K101A CsoRs reveals that these two group IV clade-specific residues contribute to apoprotein DNA binding affinity, with K101A CsoR essentially inactive under these conditions (Fig.…”

Functional and Expression Analysis of the Metal-Inducible dmeRF System from Rhizobium leguminosarum bv. viciae

“…For instance, an E34Q mutation in RcnR yielded a higher response to cobalt (45). Also, recent evidence indicates that RcnR His-67 is involved in the interaction of RcnR with cobalt (20). These two residues are not conserved in the case of R. leguminosarum dmeR.…”

“…Alignment of the two proteins revealed that the R. leguminosarum gene product contained a conserved cysteine residue (Cys-35) and three out of the four conserved histidine residues (His-3, His-60, and His-64) involved in response to Ni(II) and Co(II) (20,45); furthermore, structural prediction based on I-TASSER software (46) indicated the presence of three alpha helices similar to those present in CsoR/RcnR (see Fig. S1).…”

“…E. coli RcnR represses the expression of the Ni-and Co-specific efflux system RcnAB by binding to a specific sequence with G/C tracts flanked by AT-rich inverted repeats in the operator region (19). This repression is released upon binding of Ni or Co ions to a histidine-rich motif (H-C-H-H) critical for RcnR function (20).…”

Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

“…47, 48 It has been previously suggested that metal/small molecule binding residues can be identified in members of the CsoR/RcnR family of transcriptional regulators by utilizing a W-X-Y-Z fingerprint motif. 37, 49 In Ec RcnR, these positions are filled by His3, Cys35, His60, and His64, although His3 is not a Ni(II) ligand.…”