C‐terminal Cysteines of CueR Act as Auxiliary Metal Site Ligands upon HgII Binding—A Mechanism To Prevent Transcriptional Activation by Divalent Metal Ions?

Abstract Intracellular CuI is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. It is intriguing that HgII does not activate transcription, as bis‐thiolate metal sites exhibit high affinity for HgII. Here the binding of HgII to CueR and a truncated variant, ΔC7‐CueR, without the last 7 amino acids at the C‐terminus including a conserved CCHH motif is explored. ESI‐MS demonstrates that up to two HgII bind to CueR, while ΔC7‐CueR accommodates only one HgII. 199mHg PAC and UV absorption spectroscopy indicate HgS2 structure at both the functional and the CCHH metal site. However, at sub‐equimolar concentrations of HgII at pH 8.0, the metal binding site displays an equilibrium between HgS2 and HgS3, involving cysteines from both sites. We hypothesize that the C‐terminal CCHH motif provides auxiliary ligands that coordinate to HgII and thereby prevents activation of transcription.

Abstract: Intracellular Cu I is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. It is intriguing that Hg II does not activate transcription,a sb is-thiolate metal sites exhibit high affinity for Hg II .H ere the binding of Hg II to CueR and at runcated variant, DC7-CueR, without the last 7a mino acids at the C-terminus including a conserved CCHH motif is explored. ESI-MS demonstrates that up to two Hg II bind to CueR, while DC7-CueR accommodates only one Hg II . 199m Hg PACa nd UV absorption spectroscopy indicateH gS 2 structurea tb oth the functional and the CCHH metal site. However,a ts ub-equimolar concentrationso fH g II at pH 8.0, the metal bindings ite displays an equilibrium between HgS 2 and HgS 3 ,i nvolving cysteines from both sites. We hypothesizet hat the C-terminal CCHH motif provides auxiliary ligandst hat coordinate to Hg II and therebyp revents activation of transcription.
The CueR metalloregulatory protein controls cellular copper homeostasis by activating the transcription of cueO and copA genes in prokaryotesa nd some eukaryotes. [1] CueR responds to Cu I ,A g I and Au I ,b ut not to the divalent ions Hg II or Zn II . [2] SC-XRD studies on Escherichia coli CueR and EXAFS in solution revealed that the inducer metal ions are coordinated by C112 and C120 residues in al inear,b is-cysteinate fashion. [2,3] These two cysteines are essential to the protein function, as shown by mutation studies (C112S and/or C120S) both in vitro [3] and in vivo. [4] CueR proteins from variousb acteria contain two additional well conserved cysteines at the C-terminal, disordered segment of the protein (Figure1). [2] Crystal structures of the activator and the repressor forms of the DNA-bound CueR dimer suggest that at wo-turn helix between the metal binding loop and the CCHH motif may have ak ey role in the protein function. [5] Upon Ag I binding, the activator conformation is stabilized by the docking of the C-terminal helix (via residues I122, I123, L126) into an opened, hydrophobic pocket,formed by residues of the dimerization helix and the DNA-binding domain. This results in as mall "scissoring" movement and bending of the DNA chain allowing the transcription to be carriedo ut by the RNA polymerase. The allosteric role of the C-terminal helix was confirmed by constructing the Cu I -independentc onstitutive activator( T84V/N125L/C112S/C120S) and the constitutive repressor(truncation from I122) mutants of CueR. [5] Several representative examples can be found in the literature where non-cognate metal ions bind to am etalloprotein with the same or even higher affinity than the inducer metal ion. However,d espite the high affinity binding of non-cognate metal ions, they cannot trigger the functional structural change of the protein, because the coordination number or geometry differ. [6][7][8][9] Thus, studyingt he interaction of metalloregulatory proteins with non-cognate metal ions may provide ad eeper insight into the mechanism of metal ion selection and the regulationo ft he transcription. [8] Although CueR is one of the most thoroughly characterized proteins in the MerR family,t he mechanism of discrimination between mono-a nd divalent metal ions is still not fully understood.S urprisingly,H g II does not trigger the activation of transcriptionb yC ueR, [2] despite its well-known preference for a bis-thiolate coordination environment. [10] O'Halloran et al. determined aC u I -binding sensitivity of the CueR protein (1-2 10 À21 m)b ased on an in vitro transcriptionala ssay. [2] Ourp revious studies on model peptides of the metal bindingl oop of CueR also showedt hat these fragments bind Cu I with ah igh affinity. [11] However, accordingt om odel peptide studies [12,13] and QM/MM calculations, [14] Hg II ions may be coordinatede ven more efficiently. Moreover,H g II is also able to bind to aC Cs equence, [15] and therefore coordinationo fH g II ion by the CCHH motif is also highly probable.
With the present work we aim to explore the role of the Cterminal CCHH motif with ap articularf ocus on the bindingo f Hg II to CueR. To achievet his, we studied the Hg II -interactiono f E. coli CueR and its truncated variant, lacking seven C-terminal residues (including the CCHH motif), DC7-CueR. The integrity of this variant was confirmed by CD spectroscopy ande lectrophoretic mobility shift assay,see FigureS3.
As eries of ESI-MS spectra were recorded with the two protein variants, see Figures 2, S4 and S5. The disappearance of the signals of the apo-form in the presence of 1.0 equivalent of Hg II implies that Hg II ions display high affinity to both proteins. The spectrao btained at twofold Hg II -excess per protein clearly demonstrate the availability of two binding sites for Hg II ions in the Wild-type (WT) CueR. These are most likely the metal ion binding loop formed by C112 and C120, and the Cterminal CCHH motif. Participation of the latter CCHH sequencemotif in Hg II binding is supported by the lack of signals correspondingt oaH g 2 -DC7-CueR complex, even at twofold Hg II -excess over the truncated protein. Both the Hg-CueR and Hg 2 -CueR speciesa re observed at 1.0 equivalent Hg II ,s uggesting that there is no significant differencei nt he Hg II -binding affinities of the two sites.
199m Hg-perturbed angular correlation (PAC) spectroscopy [12,13,[16][17][18][19][20] was used to elucidate the metal site structures and dynamics at the nanosecond timescale, see Figure 3a nd Supporting Information Figure S6. At pH 6.0 andH g II :CueR of 0.2 and 1.0, the signals agree well with aH gS 2 coordination geometry,t hat is, coordination of Hg II by two cysteinates. [18] This is also the case at Hg II :CueR of 2.0, although as lightly larger linewidth is observed, in particularf or the first peak at around 1.4 rad ns À1 .T his line broadening presumably reflects the occupationo ft wo HgS 2 sites, and it can originate either from minor differences in structure of the two sites, or from metal site dynamics at the nanosecond time scale becomingm ore pronounced upon binding of the second Hg II (Figure 3).
The spectrum recorded with 0.2 equivalent of Hg II per CueR at pH 8.0 is more complex than at pH 6.0. Qualitatively,t he first peak is shifted to slightly lower frequency and exhibits considerable broadening, and the second peak (ca. 2.8 rad ns À1 )i ss ignificantly attenuated, to the extent that it barely rises above the noise level. Ar eliable analysiso ft he data requires the inclusion of two nuclear quadrupole interactions (NQIs). One of these NQIs is very similart ot hat observed in the spectra at pH 6.0, most likelyr eflecting aH gS 2 structure. The other NQI has ah igher asymmetry parameter and al ower frequency,s ee Ta ble S1, indicating ah igherc oordination number than 2. The lower frequency agrees well with an ideal trigonal planarH gS 3 structure, but the relativelyh igh asymmetry parameter rules out this possibility.H owever,i nt he simple angular overlap model (AOM), [21] aT -shaped HgS 3 coordination geometry gives the same frequency as at rigonal planar structure, but an asymmetry parameter of 1. Thus, aH gS 3 structure in betweent rigonal planar and T-shaped, with the third ligand in as lightly longer HgÀSd istance seems to be ap lausible structurali nterpretationo ft he low frequency signal. It is also  possible that the PACd ata reflect at rigonal planarH gS 2 N structure, with ah istidine coordinating, ast his would give an asymmetry parameter different from zero. However,t his seems less likely,g iven the thiophilicityo fH g II ,a nd the UV absorption data, vide infra. Finally,i ti sc onceivable that the spectrum reflects intermediate (nanosecond) exchange between HgS 2 and HgS 3 structures. Notice that this entails af lip of principal axis of the electric fieldg radient tensor, whichh as V zz along the axis of HgS 2 but perpendicular to the HgS 3 plane, and therefore the asymmetry parameter will depend on the dynamics in an on-trivial manner.I tc annot be excluded that the data recorded at 1.0 equivalent of Hg II also contain signals reflecting both of these species, but the reduced chi-square does not improve significantly upon including as econd NQI. Consequently, we have only included the high frequency NQI (HgS 2 )i nt he analysis. For the experiment with 2.0 equivalents of Hg II the signal may be satisfactorily fitted with just one (high frequency) NQI, presumably reflecting HgS 2 structure for both Hg II bound to CueR (Figure 3).
Most interestingly, the 199m Hg PACs pectrum recorded at pH 8.0 with 0.2 equivalents Hg II for DC7-CueR exhibits as ignal reflecting only HgS 2 structure (Figure 3). The fact that the DC7-CueR Hg II site exhibits aH gS 2 structure strongly supportst he interpretation presented above for the WT CueR:i fH gS 3 is formed by occupation of the functional site, at hird thiolate is recruited from the CCHH motif, or vice versa, Hg II binds to the CCHH motif and recruits one of the cysteines from the functional binding site. With 2.0 equivalents of Hg II per DC7-CueR at pH 8.0, the signal changes as compared to experimentsw ith 1equivalent Hg II ,p resumably because the functionalm etal site is filled, and the additional Hg II accommodates ac oordination geometry other than linear HgS 2 due to weak or non-specific Hg II adducts.T his agreesw ell with the ESI-MS data, where no Hg 2 -DC7-CueR was observed. Thus it is likely that the signal includes more than one NQI. Surprisingly,t he signal shifts to slightly higher frequency,w hich is difficult to account for, except if ap ositive charge appears in the equatorial plane of HgS 2 ,vide infra.
To further characterize the metal site coordination geometries, we appliedU Va bsorption spectroscopy ( Figure 4). Hg IIthiolatec omplexes possess characteristic charge transfer (CT) bands in the region of 230-300nm. Moreover,f eatures of the absorption spectrum reflect the coordinationg eometry of the complexes.U sing Hg(SEt) 2 3 ]m odel compounds, the UV-absorption spectra of linearly and trigonal planar coordinated Hg II ,r espectively,w ere characterized. [24] Linearly coordinated Hg II -thiolate speciesd isplay at ransition at around2 30 nm. [22] The increaseo ft he coordination number shifts the absorption bands towards longer wavelengths. [23,25] The spectrum of at rigonal Hg II -thiolate complex has ac haracteristic absorption maximum at 245 nm with ad istinct shoulder at around 290 nm. [22] Qualitatively,t he absorption difference spectra at sub-equimolar Hg II :WT CueR ratios exhibit ac haracteristic absorption at around 290 nm reflecting the presence of HgS 3 structure (Figure 4), in agreement with the PACd ata, vide supra. The PACd ata indicate 40 %H gS 3 and 60 %H gS 2 at 0.2 equivalents Hg II .W eu sed the recorded spectrumw ith 2.0 equivalents Hg II per WT CueR ( Figure 4A)t od etermine the molar absorption of the HgS 2 species (green curve in Figure 4C). Next, we predicted the pure HgS 3 molar absorption spectrum ( Figure 4C,p urple curve) by assuming that the experimentally determined spectrum is given by 0.6H gS 2 + 0.4 HgS 3 .T he UV absorption spectra derived in this manner for HgS 2 and HgS 3 agree well with those reported in the literature, [23] strongly supporting the interpretationo ft he PACd ata presented above. We presentm olar absorption data at selected wavelength values in Ta ble 1. The UV absorption spectra recorded for DC7-CueR exclusively exhibit the signatureo fH gS 2 structure, corroborating the interpretation of other experimental data. Surprisingly,t he absorbance for DC7-CueR continues to increaseb eyond1 .0 equivalentH g II and saturates at ca. 2:1 Hg II :DC7-CueR, indicatingt hat the truncated protein can accommodate two Hg II ions in aH gS 2 coordination environment. This may be realized if ad inuclear Hg 2 S 2 site is formed with the two thiolates as bridging ligands. Interestingly,t his agrees with the unexpectedly high frequency observed by PACs pectroscopy, which can be explained by the presence of ap ositive charge (the second Hg II )i nt he Hg 2 S 2 structure, vide supra. The fact that the species with two Hg II bound per CueR monomer is not observedi nE SI-MS implies that the binding of the second Hg II is relativelyw eak.

and [Et 4 N][Hg(SBut)
In Figure5,w ep resent model structures which agree with all the experimental data presented in this work. At pH 8.0 with 0.2 and 1.0 equivalent Hg II ,t wo speciesc o-exist, most likely the linear HgS 2 and aH gS 3 structure with the equatorial Hg-S bond being longert han the other two. Such structures have also been observed in small, Hg II containing inorganic compounds. [26] The increased availability of deprotonatedc ysteines with increasing pH agrees well with this change in speciationo bserved from pH 6.0 to pH 8.0, that is,achange from HgS 2 towards HgS 3 coordination mode, and as imilar trend has been observed for de novo designed proteins by Iranzo et al. [18] The additional thiolate is most likely recruited from the CCHH motif, or vice versa, and may thusp revent the docking of the C-terminal helix into the hydrophobic pocket, and consequently inhibita ctivation of transcription. The net negative chargeo fH gS 3 may be stabilized due to the presence of lysine or arginine in the C-terminal fragment of CueR in almost all the organismsl isted in Figure 1. That is, we hypothesize that the CCHH motif is not involved in the function of CueR when sensingt he monovalent coinage metals, but it does take part in binding of divalent metal ions, am echanism that would account for the selectivity of CueR.
It may seem intriguing that with 1.0 equivalent Hg II both the PACa nd UV absorptions pectra differ significantly from those recordedw ith 0.2 equivalentH g II .H owever,as imple probabilistic model qualitativelya ccounts for this change, assuming that the two sites are independent (i.e. distributing Hg II randomly among the 4m etal sites of ap rotein dimer), and that population of two adjacent sites (the functional site andt he Cterminal site) leads to formation of HgS 2 ,b ecause there are no more cysteines locally availablet of ormH gS 3 ,s ee the Supporting Information for details. This very simple interpretation is to some extents upported by the ESI-MS data, which display populationo ft he Hg 2 -CueR speciesw hen Hg II andC ueR are present in equimolar amounts. Obviously,t he model is too simple because formation of HgS 3 requires that cysteines from both metal binding sites are involved, but the alternative, that is, that one binding site (eithert he functional site or the C-terminal site) binds Hg II with significantly highera ffinity than the other, does not agree with the spectroscopicd ata, because this would imply that the HgS 2 /HgS 3 ratio should be the same at 0.2 and at 1.0 equivalent, nor with the ESI-MS data, which indicatet he presence of Hg 2 -CueR already at 1.0 equivalent Hg II .A t2 .0 equivalents Hg II ,o fc ourse,t here is no more possibility to form HgS 3 ,b ecauset he protein is saturated with Hg II in HgS 2 structures.S imilarg eometrical rearrangement was observed in metallothioneins (by UV absorption)u pon saturating the protein by the metal ion in at itration with Hg II . [27,28] The functiono ft he CCHH motif has also been studied by Stoyanov and Brown,u sing an in vivo assay to monitort he CueR controlled transcription. [4] The double mutation of histidine (H131N/H132N) or cysteineresidues (C129S/C130S)a nd truncation from G128 in E. coli CueR resulted in an only slightly altered induction of the transcription by cognate metal ions. Althoughe xperimental dataw ere not presented, Stoyanova nd Brown indicated that the selectivity of reactionw ith other,u nspecified metal ions was not affected. To furthere xplore this issue, as eries of in vitro and in vivo transcriptional assays should be conducted.  In summary,w eh ave demonstrated that up to two Hg II ions bind with high affinity to WT CueR, one at the functional (C112 and C120) metal binding site, and the other at the C-terminal CCHH motif. Moreover,u nder conditions where the protein is not saturated by Hg II ,ah igher coordinationn umber (presumably HgS 3 )i so bservedf or WT CueR but not for DC7-CueR, indicating that side chains from the CCHH motif may be recruited as auxiliary ligands at the functional metal site (or vice versa). This implies am echanism where the specificityo fC ueR for monovalent coinage metal ions and against divalent metal ions is achieved by coordination to divalent metal ionsb yt he CCHH motif, preventing the docking of the C-terminal helix into the hydrophobic pocket, [5] andc onsequently inhibiting activation of transcription. Indeed, the CCHH motif provides as election of ligands that may participate in coordination of both soft and intermediate metal ions. As the findings presented here on Hg II do represent as pecial case, the generalization to other divalent metal ions should be considered carefully.