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# Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by CueR

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Science  05 Sep 2003:
Vol. 301, Issue 5638, pp. 1383-1387
DOI: 10.1126/science.1085950

## Abstract

The earliest of a series of copper efflux genes in Escherichia coli are controlled by CueR, a member of the MerR family of transcriptional activators. Thermodynamic calibration of CueR reveals a zeptomolar (1021 molar) sensitivity to free Cu+, which is far less than one atom per cell. Atomic details of this extraordinary sensitivity and selectivity for +1transition-metal ions are revealed by comparing the crystal structures of CueR and a Zn2+-sensing homolog, ZntR. An unusual buried metal-receptor site in CueR restricts the metal to a linear, two-coordinate geometry and uses helix-dipole and hydrogen-bonding interactions to enhance metal binding. This binding mode is rare among metalloproteins but well suited for an ultrasensitive genetic switch.

E. coli maintain a strict cellular copper quota within a narrow range, about 104 atoms per cell (∼10 μM), by using numerous copper homeostasis pathways to control and allocate the metal to a few important enzymes (1, 2). One of the first responses of E. coli to even mild copper stress is the expression of the efflux pump CopA, a homolog of the Menkes and Wilson disease proteins, which removes Cu+ from the cytosol into the periplasm (3, 4). Copper-induced expression of CopA and CueO, an oxidase postulated to catalyze the air oxidation of Cu+ to the less toxic Cu2+ form in the periplasm (5, 6), is controlled by CueR (3, 79), a member of the MerR family of metalloregulatory proteins. This family includes metal-responsive transcriptional activators such as the zinc sensor ZntR (1, 10) and the mercury sensor MerR (11).

Similar to other MerR metalloregulatory family members, CueR exhibits a metal-selective behavior in vivo, activating the transcription of copA in response to elevated extracellular concentrations of the salts of coinage metals, such as copper, silver, and gold, but not of zinc and mercury (79, 12). Such selectivity can derive either from cellular components (i.e., metallochaperones or transporters) that control metal accessibility to CueR or from the intrinsic metal-recognition properties of CueR itself. To address the molecular basis of this selectivity, we thermodynamically calibrated CueR in an in vitro metal-responsive transcriptional switching assay using well-defined copper-metal–buffering systems. Various metal salts were titrated into run-off transcription assays, in which purified CueR regulates RNA polymerase (RNAP) transcription at the PcopA promoter (13). Initial studies of CueR show a lack of metal-dependent transcriptional activation: The switch is “on” without addition of any metal ions, even in the presence of high concentrations of avid Cu+-binding ligands such as glutathione (GSH) (14, 15) (fig. S1). Transcriptional activation by CueR is suppressed only when millimolar concentrations of cyanide (CN), which has a higher affinity for Cu+ than has glutathione (14, 16), are added to the assays. Transcription can be recovered upon the addition of Cu+ back to the cyanide-containing assays, thus indicating that copper binding to CueR is tight but reversible under these conditions.

Titration of different metals in the presence of 1.0 mM cyanide (Fig. 1A) reveals transcriptional-activation profiles that reproduce the in vivo behavior of the CueR/promoter system (7, 9, 12). Transcript levels rise steadily with increasing amounts of Cu+, reaching saturation at 50 μM total Cu+. Titrations with either Ag+ or Au+ ions in the presence of 1.0 mM CN gave similar CueR-response profiles (Fig. 1A). Consistent with in vivo results (9), neither Hg2+ nor Zn2+ induced any CueR-mediated transcription in vitro (Fig. 1A). Indeed, little direct binding of Hg2+ or Zn2+ to CueR is observed when competitors such as dithiothreitol (DTT) are present (17). The parallels between the in vivo and the in vitro studies indicate that CueR is capable of directly distinguishing metal ions with a +1 charge from metal ions with a +2 charge in gene regulation.

To calibrate the Cu+ response profile of CueR, we designed additional transcription assays under a series of metal-buffering conditions that precisely control the free Cu+ concentration. Given critical stability constants for the Cu+/CN system, the pKa of CN (where Ka is the acid dissociation constant), and total CN concentrations that exceed both copper and protein concentrations (i.e., ≥1.0mM), the free Cu+ concentration in vitro can be readily buffered at values in the range of 1023 to 1018 M (pH 8.0) (fig. S2) (16). Half-maximal CueR induction occurs at a free Cu+ concentration of 2 × 1021 ± 1 × 1021 M, corresponding to a zeptomolar CueR sensitivity for Cu+ (Fig. 1B). For comparison, the Zn2+ sensor, ZntR, exhibits half-maximal induction at a much higher level (1.15 × 1015 M free Zn2+ concentration) (1). Considering that the lowest formal intracellular concentration of free copper is ∼109 M (i.e., one free copper atom per E. coli cell with a volume of 1.5 × 1015 liter) (1), the CueR switch is tripped at a copper concentration that is formally 11 orders of magnitude lower than one free Cu+ atom per cell. This thermodynamic result indicates that Cu+ binds to CueR much more tightly than to glutathione, the most abundant thiol in the cell, and is consistent with the observation that GSH alone is not a reasonable competitor for Cu+ in the CueR activation of PcopA (fig. S1). These results lead us to propose that neither free Cu+ nor copper-glutathione complexes constitute a persistent “copper pool” in the prokaryotic cytosol under normal growth conditions.

To understand metal sensitivity and selectivity in the MerR family of metalloregulatory proteins at the atomic level, we solved the x-ray crystal structures of metal-bound forms of two representative members that discriminate between metal ions with a +1 and a +2 charge: E. coli CueR and E. coli ZntR. The structures of Cu+-, Ag+-, and Au+-bound forms of CueR were determined to 2.2, 2.1, and 2.5 Å resolution, respectively, whereas the structure of an N-terminally truncated fragment of ZntR bound to Zn2+ was solved to 1.9 Å resolution (13). The overall structure of the CueR dimer is identical in all three metal-bound states. Each monomer can be divided into three distinct functional domains: a dimerization domain flanked by a DNA-binding domain and a metal-binding domain (Fig. 2A). The structure of the CueR DNA-binding and dimerization domains is characteristic of the MerR family of proteins and shares the same topology as the structures of two other MerR-family transcriptional activators, BmrR (18) and MtaN (19, 20), which respond to the presence of organic substrates.

In each CueR structure, the metal ion is buried in a solvent-inaccessible site in a loop at the dimer interface (Fig. 2B) and has only two coordinating ligands: the S-atoms of conserved Cys112 and Cys120 (Fig. 3A). These residues define the end points of a 10-residue loop that extends from the C-terminal end of the dimerization helix up to the N-terminus of a short two-turn α helix. In the dimer, only one metal-binding loop is fully ordered, even though another metal is bound at the equivalent site in the dimer. Residues 115 to 119 of the second monomer are disordered, perhaps due to differences in crystal packing. This disorder suggests that flexibility in the extended region of the metal-binding loop may allow metal access to the buried binding site. The metal-binding loop of one monomer rests against the N-terminal end of the long dimerization helix and the preceding loop of the other monomer, interacting primarily through backbone contacts. The short two-turn α helix that extends from the metal-binding loop packs against the DNA-binding domain of the other monomer and against both dimerization helices by way of several conserved hydrophobic residues (Fig. 3). These extensive hydrophobic interactions effectively form a scaffold that stabilizes the buried metal-binding site.

Coordinate-covalent bonds between the Cu+ ion and the two sulfur atoms of Cys112 and Cys120 exhibit Cu-S bond distances of 2.13 Å with an essentially linear S-Cu-S bond angle of 176° (Fig. 3A). Extended x-ray absorption fine structure studies on copper-CueR complexes confirm this coordination environment and demonstrate that the copper is unequivocally in the +1 oxidation state (21). The metal-binding domain in Ag+-CueR and Au+-CueR is identical to that found in Cu+-CueR, except for longer metal-sulfur bond distances (Ag-S: 2.35 Å; Au-S: 2.32 and 2.39 Å). All sulfur-to-metal distances and bond angles (176° to 177°) are consistent with the average values for linear, two-coordinate Cu+, Ag+, and Au+ complexes found in the small-molecule Cambridge Structural Database.

Mutation of Cys112 or Cys120 in CueR abrogates all response to Cu+, Ag+, and Au+ in vivo (12) and in vitro (21), and the structure confirms that the surrounding environment does not allow for other types of interactions with the metal. The closest residue with metal-binding potential is Ser77 from the second monomer, which has a Cu-Oγ distance (4.4 Å) that is too long for primary or secondary covalent-bonding interactions. Instead, Ser77 is within hydrogen-bonding distance to Asp115 and several main-chain atoms of the metal-binding loop (Fig. 3, A and B). These interactions may be important in maintaining the conformation of the metal-binding loop and stabilizing the quaternary interactions in the dimer interface.

The overall structure of the truncated ZntR dimer is similar to that of CueR and consists of one helix-turn-helix motif of the DNA-binding domain, the dimerization helix, and an intact metal-binding domain. In ZntR, both metal-binding domains are well ordered with two Zn2+ ions bound at each site through Cys114, Cys115, His119, Cys124, and Cys79 (Fig. 3A), which is supported by biochemical data showing a diminished response to Zn2+ upon mutation of any of these residues (22). The dinuclear Zn-binding site shows each Zn2+ ion in a tetrahedral coordination environment with a Zn-Zn distance of 3.6 Å. Cys114 and Cys124 (equivalent to Cys112 and Cys120 in CueR) serve as ligands to Zn1, whereas Cys115 and His119 coordinate Zn2. Cys79 (equivalent to Ser77 in CueR) from the other monomer acts as a bridging ligand to the two Zn2+ ions, thus linking the metal-binding domain of one monomer and the dimerization domain of the other monomer. An oxygen atom of a bridging phosphate or sulfate group acts as a fourth ligand to each Zn2+ ion. In contrast to the CueR metal-binding site, the coordination environment of the metal-binding loop in ZntR is optimal for binding of divalent metals.

The crystal structures of CueR and ZntR reveal several determinants of metal-ion selectivity. A residue at the N-terminus of the dimerization helix extends across the dimer interface to contact either the metal-binding loop (Ser77 in CueR) or directly to the metal ion (Cys79 in ZntR) and plays a unique role in discriminating between +1 and +2 ions (Fig. 3). A serine is found at this position in all MerR homologs responsive to +1 ions, whereas a cysteine is present in homologs that are known to respond to +2 ions (Fig. 4). Because +2 metal ions typically prefer higher coordination numbers than +1 ions of the coinage metals, discrimination is first conferred at the level of coordinate-covalent bond formation. Second, CueR restricts the bound metal to a low coordination number by hydrophobic and steric restrictions, which contribute to a shielded coordination environment.

Electrostatic features of the metal receptor cavity are the third contributing factor in defining the extraordinary metal affinity and selectivity. In CueR, the buried S-Cu-S center formally has a net negative charge arising from two thiolate anions and one +1 charged metal. The structure reveals a series of weak interactions that compensate this buried charge and provide an electrostatic component to the metal-binding free energy beyond the Cu-S bond energy (Fig. 3, B and C). Charge neutralization of the thiolate of Cys120 likely arises from interactions with the positively charged end of the helix dipole and two backbone H bonds that originate from the short two-turn α helix extending from the metal-binding loop. In CueR, Pro121 constrains the S atom of Cys120 such that it is centered directly over the N-terminus of the short helix (Fig. 3C). A Cys-Pro motif at the N-terminus of an α helix allows for favorable interactions of the cysteine with the helix dipole (23), and such a motif is conserved at the end of the metal-binding loop in many of the MerR family members (Fig. 4). Mutation of this proline diminishes the Hg2+ response of MerR, consistent with a key role for the Cys-Pro motif (24). Cys112 accepts only one hydrogen bond (Fig. 3B) and the closest positively charged residue is a conserved lysine, Lys81 (the Nζ-S distance is 5.5 Å). The charge-charge interaction of Lys81 with the Cys112 thiolate, although distant, could contribute additional charge neutralization. These electrostatic- and hydrogen-bonding interactions lead to charge neutrality when a +1 ion, but not a +2 ion, binds. This allows CueR to discriminate against Hg2+-binding in what is otherwise a stable linear dithiolate Hg-coordination environment.

A structure-based sequence alignment of MerR metalloregulatory homologs allows for predictions of metal selectivity in other family members (Fig. 4). A serine or cysteine at the N-terminal end of the dimerization helix provides a clear distinction between putative +1 and +2 metal-responsive subgroups within the MerR family. In the case of PmtR, a role in Zn2+ homeostasis has been proposed (25); however, the structure-based alignment (Fig. 4) suggests a role in Cu+ regulation. For SoxR, a MerR homolog that uses an Fe-S cluster to sense oxidative stress (26), the alignment suggests that the 2Fe-2S cluster will bind in a loop resembling the ZntR binuclear Zn2+ site. Furthermore, the ZntR structure reveals how the prototypical metalloregulatory protein in this family, MerR (27), could bind Hg2+ using a trigonal metal-thiolate coordination environment (28) involving two cysteines from one monomer and a third cysteine, the equivalent to Cys79 in ZntR, from the other monomer (24, 2931).

The combination of thermodynamic and structural studies reveals how metalloregulatory proteins can select among +1 and +2 transition-metal ions. The extraordinary copper sensitivity of CueR suggests that the prokaryotic cytoplasm operates under conditions of copper deprivation. Given the absence of known copper chaperones in E. coli, these results raise the question of how bacterial copper-dependent proteins obtain their cofactor. All copper-dependent enzymes known to date in E. coli are found in the cell envelope (3, 3236). The answer may be that available cytosolic copper ions, bound or free, are detected by Cu+ sensors and rapidly transported to the cell envelope for incorporation or ejection. In this respect, copper homeostasis in Gram-negative bacteria shows a striking parallel to eukaryotic copper trafficking pathways. Both use a family of homologous copper-specific P-type adenosine triphosphatases to clear rapidly any available copper ions from the cytosol into more specialized compartments: the cell envelope in prokaryotes and the trans-Golgi secretory pathway in eukaryotes (37).

Supporting Online Material

Materials and Methods

Figs. S1 and S2

Table S1

References

## References and Notes

1. The Cu+ binding constants (βn, where n ≥ 2) and the proton association constant (K) of CN at 25°C and 0.1 M ionic strength were obtained from the NIST Critical Stability Constants of Metal Complexes (14): $Math$(1) $Math$(2) $Math$(3) $Math$(4) At a given total Cu+ concentration, the free Cu+ concentration at pH 8.0 was calculated with [CN]total, [Cu+]total, and the above constants using the HySS program (40) (fig. S2). Considering the metal-buffering capacity of a given ligand concentration, only the free Cu+ concentration ranges that can be precisely controlled by the chosen ligand system (fig. S2, highlighted in gray) were used in the experiments shown in Fig. 1B.
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