Dissecting the Metal Selectivity of MerR Monovalent Metal Ion Sensors in Salmonella

Abstract

Two homologous transcription factors, CueR and GolS, that belong to the MerR metalloregulatory family are responsible for Salmonella Cu and Au sensing and resistance, respectively. They share similarities not only in their sequences, but also in their target transcription binding sites. While CueR responds similarly to Au, Ag, or Cu to induce the expression of its target genes, GolS shows higher activation by Au than by Ag or Cu. We showed that the ability of GolS to distinguish Au from Cu resides in the metal-binding loop motif. Here, we identify the amino acids within the motif that determine in vivo metal selectivity. We show that residues at positions 113 and 118 within the metal-binding loop are the main contributors to metal selectivity. The presence of a Pro residue at position 113 favors the detection of Cu, while the presence of Pro at position 118 disfavors it. Our results highlight the molecular bases that allow these regulators to coordinate the correct metal ion directing the response to a particular metal injury.T ransition metal homeostasis influences many fundamental aspects of bacterial cell physiology and pathogenesis (1-3). The intracellular concentration of essential metals or the presence of harmful elements is monitored by a set of transcriptional regulators that modulate the expression of factors that rapidly restore metal homeostasis (4, 5). A large class of these metalloregulators belongs to the MerR family, a group of proteins that share similarity at the N-terminal DNA-binding domain (6-9). According to the current model, dimeric metal-sensing MerR regulators control gene transcription via a DNA distortion mechanism. Both the apo-and the metal-bound regulator recognize and interact with their target operators (a dyad-symmetric DNA sequence at the promoter region of their target genes). Binding of the metal ion at the C-terminal inductor-binding site would provoke an allosteric change at the N-terminal DNA binding region of the protein, which in turn transduces changes in the promoter structure resulting in transcription activation of the expression of genes coding mostly for efflux or detoxification systems (10-12).Most of the metal ion sensors of the MerR family are poorly selective because they cannot distinguish between cognate metals with similar physicochemical properties, including charge and coordination chemistry. For example, the Cu sensor CueR can discriminate between metal ions with ϩ1 and ϩ2 charges, but it cannot distinguish between monovalent metal ions of group 1B-i.e., Cu(I), Ag(I), and Au(I) (13). Structural studies indicate that CueR has only two coordinating ligands, the S-atoms of the conserved C112 and C120 residues which are appropriate for the interaction with the ϩ1 metal ion in a linear array but not to coordinate metal ions with a ϩ2 charge, which requires higher number of ligands (14). The recent identification of two Au-selective MerR sensors, first in the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and then in Cupriavidus met...

A Single Serine Residue Determines Selectivity to Monovalent Metal Ions in Metalloregulators of the MerR Family

“…GolS or CueR mutants were constructed basically as previously described (12). To generate the different golS alleles, a megaprimer was produced using the golS-F forward primer (5=-ATGAGGAGGAGCGTCATGAACATCG-3=) and reverse primers carrying each mutation (primer sequences are available on request).…”

“…To generate the golB::lacZ transcriptional fusion, the lac fusion plasmid pKG136 (Table 1) was inserted at the remaining FLP recombination target (FRT) site generated after removing the chloramphenicol resistance cassette as previously described (12). To generate GolS or CueR variant expression plasmids, each golS or cueR mutant allele was PCR amplified from the chromosome of the mutant Salmonella strain, using the golS-ORF-F/golS-ORF-R or cueR-F/cueR-R primer pair (10,18), and cloned into BamHI-HindIII-digested pUH21-2 laqI q (19).…”

“…By domain-swapping and site-directed mutagenesis, we established that residues at positions 113 and 118 within the MBL are the main contributors to Au(I) selectivity (12). The presence of a Pro residue at position 113 favors the detection of Cu, while the presence of Pro at position 118 disfavors it.…”

“…The presence of a Pro residue at position 113 favors the detection of Cu, while the presence of Pro at position 118 disfavors it. GolS xenologs harbor alanine and proline at positions 113 and 118, respectively, while CueR and its homologs harbor P113 and A118 instead (12). All these sensors are activated by ϩ1 metal ions but do not respond to ϩ2 ions, such as Zn(II), Cd(II), Hg(II), or Pb(II), that act as inducers of other members of the metal-responsive MerR family (2,6,14).…”

“…Two homologous monovalent metal ion sensors that coexist in Salmonella enterica were characterized: the nonselective CueR sensor, which responds to Cu(I), Ag(I), and Au(I) with similar sensitivities, and the Au sensor GolS, which discriminates Au(I) from Cu(I) or Ag(I) (10)(11)(12)(13). By domain-swapping and site-directed mutagenesis, we established that residues at positions 113 and 118 within the MBL are the main contributors to Au(I) selectivity (12).…”