Communication pubs.acs.org/IC
Structural Basis for the Selective Pb(II) Recognition of Metalloregulatory Protein PbrR691 Shanqing Huang,† Xichun Liu,† Dan Wang,† Weizhong Chen,† Qingyuan Hu,† Tianbiao Wei,† Wenquan Zhou,‡ Jianhua Gan,§ and Hao Chen*,† †
Coordination Chemistry Institute and the State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Collaborative Innovation Center of Chemistry for Life Sciences, and ‡Jinling Hospital, Department of Urology, Medical School of Nanjing University, Nanjing University, Nanjing 210093, P. R. China § School of Life Sciences, Fudan University, Shanghai 200433, P. R. China S Supporting Information *
10−14, which matches the high sensitivity of the PbrR691 protein toward Pb(II) in vivo.11
ABSTRACT: The transcription regulator PbrR691, one of the MerR family proteins, shows extremely high sensitivity and selectivity toward Pb(II) in Ralstonia metallidurans CH34. Here, we present the crystal structure of PbrR691 in complex with Pb(II) at 2.0 Å resolution. The Pb(II) coordinates with three conserved cysteines and adopts a unique trigonal-pyramidal (hemidirected) geometry. To our knowledge, the PbrR691-Pb(II) structure provides the first three-dimensional visualization of a functional hemidirected lead(II) thiolate coordinate geometry in a protein.
S
ince the metalloprotein MerR, the transcriptional factor regulating mercury resistance, was first identified in Gramnegative bacteria 30 years ago,1,2 a series of paralogous regulators that respond to various metal ions or small organic molecules with high sensitivity and selectivity have been successively characterized in a wide range of bacterial genera. The MerR family proteins include the mercury sensor MerR,3,4 copper sensor CueR,5 zinc sensor ZntR,5 multidrug transporter BmrR,6 and oxidative stress responder SoxR.7 All of these MerR-like regulators share high sequence similarity, especially in the Nterminal DNA-binding domain. The varied C-terminal regions are responsible for the specific effector recognition.8 The Pb(II) transcription factor PbrR691 [deposited in the Protein Data Bank (PDB) under accession code 5GPE] belongs to the MerR family, and it is encoded on two endogenous megaplasmids, pMOL28 and pMOL30, from Ralstonia metallidurans CH34.9,10 This protein functions as a homodimer in solution and tightly controls the unique pbr transcription operon, which has combined functions in the uptake, efflux, and accumulation of Pb(II).11 PbrR691 and its homologues are the only known metalloproteins characterized in nature that specifically respond to Pb(II).12−14 As reported by He and coworkers,15 PbrR691 could detect Pb(II) with at least 1000-fold selectivity over other metal ions, such as Cu(II), Zn(II), Co(II), Hg(II), and Cd(II). To further elucidate this Pb(II) hypersensitivity, we measured the binding affinity of Pb(II) to PbrR691 by UV−vis titration under the competition of ethylenediaminetetraacetic acid (EDTA) in vitro (Figure 1). The results show that the dissociation constant Kd is 8.1 (±0.8) × © XXXX American Chemical Society
Figure 1. Pb(II) titration of wild-type PbrR691. Optical absorption spectrum of 38 μM PbrR691 and 50 μM EDTA with increasing concentrations of Pb(II) (mangenta curves). The figure was generated by Origin8, which imported the raw data from the UV−vis spectra. Some absorption spectra have been omitted for clarity. The spectral data (absorption results at 337 nm) were corrected by subtracting the spectrum at 337 nm of 38 μM apo-PbrR691 with 50 μM EDTA (black curve). These data were used in DynaFit with an identical binding model of one site, and DynaFit calculated the dissociation constant.
As revealed by the sequence alignment and structural analysis of MerR family proteins (MerR, CueR, and ZntR),3,5 PbrR691 contains three conserved cysteine residues, Cys78, Cys113, and Cys122, which may be involved in the Pb(II) binding (Figure S1). There is no doubt that cysteines can bind heavy-metal ions (Pb2+, Hg2+, Zn2+, Cd2+, Fe2+/3+, As3+, Cu+, and Ag+) with high affinity,16,17 but why PbrR691 possesses such an unprecedented Pb(II) ion selectivity and sensitivity is still not fully understood. To elucidate the Pb(II) coordination structure in PbrR691, an EXAFS experiment was executed by the He group. However, it is not clear whether the PbrR691 protein adopts a hemidirected geometry or another classical tetrahedral structure with a Received: October 6, 2016
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DOI: 10.1021/acs.inorgchem.6b02397 Inorg. Chem. XXXX, XXX, XXX−XXX
Communication
Inorganic Chemistry possible fourth ligand in Pb(II) binding.12 Herein, we present a three-dimensional crystal structure of PbrR691 in complex with Pb(II). It is referred to as PbrR691-Pb(II) hereafter. To our knowledge, it is the first visualization of a hemidirected lead(II) thiolate structure in metalloproteins. The structure of PbrR691-Pb(II) belongs to the P1 space group and was refined to a resolution of 2.0 Å (Table S1). There are eight PbrR691 protomers per asymmetric unit; these PbrR691 protomers can be divided into four functional homodimers. As revealed by the extremely low root-meansquare-deviation values (∼0.05 Å2), the overall conformations of the four dimers are virtually identical with each other (Figure S2a,b). The overall structure of PbrR691-Pb(II) is also similar to those of other MerR family proteins. The structure contains three distinct functional domains, including the N-terminal DNA-binding domain (residues 1−78, α1-α2-β1-β2-α3-α4), the dimerization helix (residues 79−112, α5), and the C-terminal metal-binding domain (residues 113−132, η1−α6) (Figure 2).
valence electrons and one lone pair of electrons (AX3E) are predicted to have a trigonal-pyramidal stereoscopic geometry (Figure 3a).18 In the Pb(II)-S3 coordination of PbrR691, the
Figure 3. (a) Trigonal-pyramidal stereoscopic geometry of AX3E, which contains three pairs of valence electrons and one lone pair of electrons. A is the central atom, X is a coordinated ligand, and E is a lone pair (L.P.) of electrons. (b) Coordination of Pb(II) observed in the PbrR691Pb(II) structure. The Pb(II) ion is shown as a red sphere. The coordinating cysteines are shown as sticks, with S atoms colored in yellow. The 2Fo − Fc electron density is contoured at the 2σ level.
stereochemically active lone pair (SALP) is formed by hybridization of the s and p orbitals of Pb(II).19 A space-filling model of the Pb(II)-binding site in PbrR691 clearly reveals a void in the distribution of coordinated ligands (Figure S5); the existence of the void is usually taken as suggestive evidence for a SALP.19−22 The lone pair in the equatorial site takes up more space and has a larger electron density than a bonding pair, and it repels other electron pairs more strongly. Therefore, the coordinated ligands are occupied on one side of the Pb(II). Three Pb(II)−S bonds are observed from the electron density, which directly shows a trigonal-pyramidal (hemidirected) structure (Figure 3b). The three coordinated ligands are located on the same side as residue Cys78′ from one subunit and the residues Cys113 and Cys122 from the other subunit. Residue Cys78′ is located on the loop that connects the DNA-binding domain and the dimerization helix. Cys113 and Cys122 from the other monomer are located on the metal-binding loop (Figure S6). The bond angle between each Pb(II)−S is approximately 90° (Figure S7), which is much smaller than the standard tetrahedral angle (109.5°).23 Several factors may account for the small bond angles. First, the S−Pb(II)−S angle is decreased obviously because of the large repulsive force between the SALP at the equatorial site and the bonding pairs.24 Second, the coordinated S atoms are more electronegative than the central Pb atom; consequently, the electron density of the bonding pair is concentrated close to the coordinated ligands, and the bond angles are predicted to be smaller.25,26 Third, the large size of the central Pb atom distorts the molecular geometry, which can also reduce the bond angles.24 In addition, with the assistant of Arg117 of the metal-binding loop, helices α6, α4′, and α5′ constitute a binding pocket, which shields the lead(II) thiolate center from the solvent; this binding pocket may further stabilize the hemidirected configuration (Figure S8a,b). The binding affinity of R117A mutant to Pb(II) is 2.5-fold weaker than that of wild-type PbrR691 (Figure S9). Arising from the three Pb(II)−S bonds, this triliganded structure has a net negative charge, which is similar to the model of CueR.5 Several amino acids of the metal-binding loop region (Arg117 and Gly123) and the neighboring α-helix (α6; Ile124 and Leu125) form a series of van der Waals interactions, which can compensate for the buried negative charge (Figure S10). These electrostatic components
Figure 2. Cartoon representation of the structure of the PbrR691-Pb(II) dimer. The functional dimer contains two monomers, which are shown in mangenta and cyan, respectively. Pb(II) ions are shown as red spheres. There are six α-helices (α1−α6) and two β-strands in one monomer, which are labeled in the figure.
Except residue Ser119 on one molecule, other residues are welldefined for the whole structure. The N-terminal DNA-binding domain is composed of two helix-turn-helix motifs, which are connected by a pair of antiparallel β-stands (β1 and β2; Figure S3a,b). The DNA-binding domain is linked to the C-terminal metal-binding domain through a long helix (α5). The α5 forms an antiparallel coiled-coil packing interaction with the α5′ of another PbrR691 monomer; such an interaction is essential for PbrR691 to form a functional homodimer.7 The assembly of the two Pb(II)-binding sites is strictly equivalent within the dimer (Figure 2). The metal-binding region of one protomer packs against the DNA-binding domain and dimerization helix of the partner protomer. The backbone electron density clearly shows the rigidity helices (α5 and α5′) and flexibility loop (α5-α6 and α4′-α5′) around the metal-binding region (Figure S4). The crystal structure of PbrR691-Pb(II) shows that PbrR691 adopts the Pb(II)-S3 structure with hemidirected geometry, via the residues Cys78′, Cys113, and Cys122 coordinating with Pb(II). According to the valence-shell electron-pair repulsion model (VSEPR), molecules or ions that contain three pairs of B
DOI: 10.1021/acs.inorgchem.6b02397 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry and weak interactions also contribute to the hemidirected configuration. As deposited in the Cambridge Structural Database (CSD), an inorganic molecule, such as [(C6H5)4AS][Pb(SC6H5)3], also has small bond angles.27 Moreover, the average Pb(II)−S bond distance in the PbrR691-Pb(II) structure was approximately 2.7 Å, which is consistent with the molecular structure {[TmPh]Pb}(ClO4)28 (Figure S11). Crystallographic studies and ab initio analysis verified that the hemidirected coordinate geometry is favored for Pb(II) at low coordination number (2−5), whereas holodirected coordinate geometry (bonds to ligand atoms directed throughout the surface) often appears at high coordination number (9 and 10).19 Mononuclear hemidirected Pb(II) complexes with all S coordination spheres are rare in the CSD28 and PDB (Table S2 and Figure S12). However, PbrR691, the most selective metalloprotein for Pb(II) in nature, uses this unique hemidirected structure to selectively recognize Pb(II), through three conserved cysteines. Previous studies have revealed that the SALP and large ion size of Pb(II) in hemidirected complexes are crucial for this selectivity.17 Metal ions that do not have a lone pair, such as Zn(II) and Cd(II), binding predominantly in a fourcoordinated mode in proteins and peptides.17,29 Thus, other metal ions would pay high energetic penalties to enter this triligand-binding site in PbrR691.12 For example, in the Pb(II)-S3 model compound ({[TmPh]Pb}+), Pb(II) has a 500-fold greater affinity for trigonal S3 sites than Zn(II).28 Although the three cysteines in PbrR691 are paralogous to MerR (Figure S1), the environments of the metal-binding motifs in these two proteins are different, especially the residues in the metal-binding loop. The dissociation constant (Kd) of Tn501 MerR for Hg(II) was estimated to be 10−47.4,30,31 However, the value of Kd between PbrR691 and Hg(II) is 5.68 (±0.79) × 10−7, which is measured by isothermal titration calorimetry (Figure S13a−c). This means that PbrR691 binds Hg(II) almost 107-fold weakly than Pb(II). Thus, the folding of the metal-binding domain to preorganize coordination geometries in MerR family proteins is essential for selective metal-ion recognition.32 In PbrR691, the metal-binding domain also provides appropriate surroundings to form and stabilize the trigonal-pyramidal geometry, which is suited for PbrR691 to selectively recognize Pb(II) and to discriminate other soft metal ions (Figures S8a,b and S10). In conclusion, structural studies indicate that the metalloregulatory protein PbrR691 selectively recognizes Pb(II) through its unique trigonal-pyramidal coordination geometry with three conserved cysteines. Also, the UV−vis titration results reveal that PbrR691 binds Pb(II) with high affinity in vitro. This PbrR691-Pb(II) structure provides the first view of a functional, mononuclear, hemidirected lead(II) thiolate center in a protein. According to previous studies of fluorescent Pb(II) probes15 and the Ag+-CueR-DNA structure,33 once Pb(II) is bound in this site, PbrR691 switches to an activator conformation from a repressor state (metal-free), distorting the promoter DNA and initiating downstream gene transcription (Figure S14a,b). A comparison of the different metal-binding domains in the MerR family metalloregulatory proteins shows that CueR adopts a linear geometry to recognize 1+ transition-metal ions (Cu+, Ag+, and Au+),5 MerR adopts a trigonal-planar geometry to selectively recognize Hg(II),3,4 and PbrR691 adopts a hemidirected geometry to selectively recognize Pb(II) (Figure 4). We speculate that the high selectivity exhibited by these metalloregulatory proteins not only is related to the distinct physical− chemical properties of metal ions but also relies on the protein conformation of the metal-binding domain.
Figure 4. Comparison of the metal-binding domains of various MerR family proteins.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02397. Experimental procedures, X-ray data and refinement statistics, and additional tables and figures (PDF) X-ray crystallographic data in CIF format (CIF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Shanqing Huang: 0000-0003-0504-9447 Hao Chen: 0000-0001-8937-4946 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from the Ministry of Science and Technology of the China Key Project (Grants 2012CB933802 and 2015CB856303) and the National Natural Science Foundation of China (Grants 20721002 and 91013009) is gratefully acknowledged. We thank the staff of Beamline BL17U at Shanghai Synchrotron Radiation Facility, located in Shanghai, People’s Republic of China, for assistance during data collection.
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REFERENCES
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