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J. Biol. Chem., Vol. 280, Issue 40, 33716-33724, October 7, 2005

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Selective Metal Binding to a Membrane-embedded Aspartate in the Escherichia coli Metal Transporter YiiP (FieF)^*

From the Department of Biology, Brookhaven National Laboratory, Upton, New York 11973

Received for publication, June 3, 2005 , and in revised form, July 26, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The cation diffusion facilitators (CDF) are a ubiquitous family of metal transporters that play important roles in homeostasis of a wide range of divalent metal cations. Molecular identities of substrate-binding sites and their metal selectivity in the CDF family are thus far unknown. By using isothermal titration calorimetry and stopped-flow spectrofluorometry, we directly examined metal binding to a highly conserved aspartate in the Escherichia coli CDF transporter YiiP (FieF). A D157A mutation abolished a Cd²⁺-binding site and impaired the corresponding Cd²⁺ transport. In contrast, substitution of Asp-157 with a cysteinyl coordination residue resulted in intact Cd²⁺ binding as well as full transport activity. A similar correlation was found for Zn²⁺ binding and transport, suggesting that Asp-157 is a metal coordination residue required for binding and transport of Cd²⁺ and Zn²⁺. The location of Asp-157 was mapped topologically to the hydrophobic core of transmembrane segment 5 (TM-5) where D157C was found partially accessible to thiol-specific labeling of maleimide polyethylene-oxide biotin. Binding of Zn²⁺ and Cd²⁺, but not Fe²⁺, Hg²⁺, Co²⁺, Ni²⁺, Mn²⁺, Ca²⁺, and Mg²⁺, protected D157C from maleimide polyethylene-oxide biotin labeling in a concentration-dependent manner. Furthermore, isothermal titration calorimetry analysis of YiiP_D157A showed no detectable change in Fe²⁺ and Hg²⁺ calorimetric titrations, indicating that Asp-157 is not a coordination residue for Fe²⁺ and Hg²⁺ binding. Our results provided direct evidence for selective binding of Zn²⁺ and Cd²⁺ for to the highly conserved Asp-157 and defined its functional role in metal transport.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Membrane transporters in the cation diffusion facilitator family are found both in eukaryotes and prokaryotes (1). This protein family of more than 400 genetically related members is characterized by a homologous hydrophobic N-terminal domain followed by a hydrophilic C-terminal domain that is variable both in sequence and length (2). Despite sequence variability, all CDF³ family members exclusively transport zinc and other divalent metal ions across the cytoplasm or organelle membranes, thus playing a variety of important roles in cellular zinc homeostasis controls (3–7). Phenotype analyses of gene deletion and complementation have demonstrated that bacterial CDF transporters are involved in cellular resistance to a broad spectrum of divalent metal cations, including Zn²⁺, Cd²⁺, Co²⁺, Mn²⁺, Ni²⁺, and Fe²⁺ (8–13). To date, molecular identities of substrate-binding sites in CDF transporters have yet to be identified, and even less is known about metal binding selectivity at a molecular level.

The Escherichia coli metal transporter YiiP (FieF) and its homolog ZitB are the two CDF proteins that were shown to function as an obligatory Zn²⁺/H⁺ antiporter (12, 14). This proton-linked antiport mechanism permits the free energy derived from the downhill H⁺ influx to be coupled to the uphill pumping of Zn²⁺ out of the cells. The kinetics of Zn²⁺/Cd²⁺ transport was described by a two-step process involving an initial binding step followed by a conformational transition that moves the metal ion across the membrane (14). The binding of metal ions to YiiP exhibited distinctive heat reactions in response to calorimetric titrations of the Zn²⁺ Cd²⁺ Hg²⁺ series, leading to thermodynamic categorization of at least one mutually competitive binding site common to Zn²⁺, Cd²⁺ and Hg²⁺, and a set of noncompetitive binding sites (15). Other than binding to group 12 metal ions, YiiP was implicated to play a role in bacterial iron detoxification, suggesting that Fe²⁺ may be an additional substrate (12). Both Zn²⁺ and Fe²⁺ are borderline soft metal ions with a similar donor preference for a combination of three protein ligand groups as follows: the cysteine thiolate, histidine imidazole, and aspartate/glutamate carboxylate (16). Phenotype analyses of two homologous CDF transporters, CzcD from Ralstonia metallidurans and ZitB from E. coli, demonstrated that mutating a highly conserved Asp residue in the putative TM-5 rendered host cells hypersensitive to zinc, probably because of a loss of zinc efflux pumping activities (17). The Asp appeared to be essential because expression of CzcD_D158E, CzcD_D158A, ZitB_D163E, and ZitB_D163A mutant proteins conferred no zinc resistance. It is not clear, however, whether this conserved CDF aspartate is directly involved in binding and transport of Zn²⁺ and/or Fe²⁺.

In the present study, we sought to establish the functional role of the equivalent Asp residue in YiiP (Asp-157) by examining the effects of D157A and D157C mutations on metal binding and transport by using direct biophysical measurements. Furthermore, Asp-157 was localized to the hydrophobic core of TM-5 to establish a structural connection with the substrate translocation pathway where metal ions are selected and transported across the membrane. Metal binding to Asp-157 was found to be highly specific, with a strict selectivity for Zn²⁺ and Cd²⁺ over Hg²⁺, Fe²⁺, and other divalent metal ions that are thought to be the frequent CDF substrates. Because Asp-157 is one of the most conserved residues in the CDF family, selective metal binding to Asp-157 has broad structural and mechanistic implications.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis—Cloning and construction of the expression plasmid pYiiP-His were described previously (15). Site-directed mutagenesis was performed using the QuickChange site-directed mutagenesis kit (Stratagene). A mutant C287S was first prepared using the pYiiP-His plasmid DNA as template and an anti-parallel pair of primers. The resultant C287S plasmid DNA served as the parent for an additional 13 mutants, each containing a single cysteine substitution mutation at positions 10, 36, 70, 107, 144, 150, 153, 155, 157, 171, 172, 174, or 177. Sequences of these mutants were verified by DNA sequencing of both strands.

Overexpression and Purification—YiiP and mutants were overexpressed with a C-terminal extension containing a thrombin cleavage site followed by six tandem histidine residues to facilitate protein purification. The expression host cells, BL21 (DE3) pLyS, were cultured in an auto-inducing medium for unattended protein overexpression (18). Cells from overnight cultures were harvested, and membrane proteins were extracted using 7% n-dodecyl--D-maltopyranoside (DDM) as described previously (15). The detergent-solubilized proteins were absorbed by three passages through a Ni²⁺-nitrilotriacetic acid superflow column (Qiagen), which was washed free of contaminants and eluted with an elevated imidazole concentration at 500 mM. The column eluate was immediately applied to a PG-10 gel filtration column (Amersham Biosciences), yielding a desalted sample that was subsequently subjected to overnight thrombin digestion (Novagen) at a ratio of 0.5 units of thrombin per mg of protein. The completeness of thrombin digestion was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis, showing a complete conversion of the His tagged to a tag-free mass species. The resultant tag-free protein was incubated with 10 mM EDTA for 30 min and then applied to an TSK 3000SW_XL size-exclusion HPLC column (TosoHaas), pre-equilibrated with a degassed HPLC buffer (20 mM HEPES, pH 7.0, 100 mM NaCl, 12.5% glycerol, 0.05% DDM, 0.2 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)). The purified protein was collected as a discrete chromatographic fraction using a Beckman SC100 fraction collector.

Isothermal Titration Calorimetry—Protein aggregates and trace amounts of metal contaminants were removed by size exclusion HPLC prior to calorimetric titrations as described previously (15). Protein concentrations were determined by BCA protein assay (Pierce). Calorimetric titrations were carried out on a Microcal MCS titration calorimeter (Microcal) at 25 °C. Metal titrants (chloride salts), typically in the concentration range of 0.25–0.5 mM, were dissolved in the same HPLC mobile phase used for protein purification. 1 mM ascorbate was added when FeCl₂ was titrated. Titrants and protein samples were thoroughly degassed, and then 30–50 injections of an indicated titrant were made successively into a protein sample in 5-µl increments at 210–360-s intervals. Heats of titrant dilutions were measured by making identical injections into the HPLC buffer, subtracted from the corresponding total heats of reaction to yield net reaction heats. The titration data were deconvoluted based on a binding model containing either one or two sets of noninteracting binding sites by a nonlinear least squares algorithm using the Microcal Origin software. The binding enthalpy change H, association constant K_a and the binding stoichiometry n were permitted to float during the least squares minimization process and were taken as the best fit values.

Reconstitution and Stopped-flow Transport Assay—HPLC-purified YiiP, YiiP_D157C, or YiiP_D157A was reconstituted into liposomes made of E. coli polar lipids (Avanti%20Polar%20Lipids">Avanti Polar Lipids) as described previously (14). Control liposomes were prepared following exactly the same procedure without adding protein. 200 µM fluorescence indicator fluozin-1 (Molecular Probes) was encapsulated by two freeze-thaw cycles, followed by gel filtration to remove the untrapped dye. Transport experiments were performed at 8 °C on a stopped-flow apparatus (KinTek Corp.). Proteoliposomes and a transport assay buffer (20 mM HEPES, 50 mM K₂SO₄, and either ZnSO₄ or CdSO₄ at an indicated concentration ranging from 0 to 4 mM, pH 7.3) were loaded into two separate syringes of equal volume, and transport reactions were initiated by pushing 60-µl fresh reactants at a 1:1 ratio through the 12-µl mixing cell at a flow rate of 20 ml/s. Zn²⁺ or Cd²⁺ concentrations in reaction mixtures were half of the concentrations in the initial transport assay buffers. Stopped-flow traces were the cumulative average of five successive recordings at 525 nm (excited at 490 nm). Liposome traces were collected as base lines and subtracted from proteoliposome traces to yield net fluorescence changes F. F/F_max was obtained by normalizing F to the maximum proteoliposome response elicited by a transport assay buffer containing 4 mM ZnSO₄ or CdSO₄ plus 2% n-octyl--D-glucoside used to solubilized proteoliposomes.

Determination of Transport Kinetic Parameters—The rate of the fluorescence rise k_obs was determined by least squares fit of the kinetic trace to a single exponential function using the data analysis software SigmaPlot 4.0 (SPSS Inc., Chicago, IL). The following two-step kinetic scheme (Scheme 1) was used to describe the YiiP transport process (14),

(SCHEME 1)

where T₁ and T₂ are different conformational states of YiiP, and M is the metal ion substrate. k₁, k₂, and k₃ are the rate constants. The dissociation constants of metal binding K_d = k₂/k₁. The relationship among k₁, k₂, and k₃ is defined as K_m = (k₂ + k₃)/k₁. Application of the steady-state condition to the species MT1 gives Equation 1 as described previously (19).

(Eq. 1)

Thus, Equation 2,

(Eq. 2)

k₃ and K_m were determined by linear regression of 1/K_obs as a function of 1/[M].

MPB Labeling—MPB labeling was carried out with cells that expressed YiiP or a YiiP variant as indicated. Cells (1 ml) from overnight cultures in the auto-inducing medium were pelleted by centrifugation and resuspended in a reaction buffer (0.5 ml) containing 20 mM HEPES, 100 mM NaCl, 2 mM MgCl₂, 10% sucrose, 0.25 mM TCEP, pH 7.5. A freshly prepared MPB stock solution (50 mM) was added to a final concentration of 2 mM, or an equal volume of double deionized water (20 µl) instead of MPB was added to a control sample as indicated. Cells were incubated with MPB at room temperature for 30 min with or without sonication (30 s), and then -ME (20 mM) was added to quench the unreacted MPB. The resulting cells were pelleted again and washed, and membrane proteins were extracted using a solubilization buffer (20 mM HEPES, 100 mM NaCl, 1% DDM, 20% glycerol, 0.25 mM TCEP, pH 7.5) with brief sonication (1 min), followed by a 30-min incubation at 10 °C to achieve complete detergent solubilization. Cellular debris was removed by centrifugation (10,000 x g for 30 min), and supernatants were collected and incubated with 20 µl of Ni²⁺-nitrilotriacetic acid superflow resin for 30 min. The resin was washed free of contaminants with 2 ml of wash buffer (20 mM HEPES, 300 mM NaCl, 40 mM imidazole, 12.5% glycerol, 0.05% DDM, and 0.25 mM TCEP, pH 7.0), and then eluted with 50 µl of elution buffer (20 mM HEPES, 100 mM NaCl, 500 mM imidazole, 12.5% glycerol, 0.05% DDM and 0.25 mM TCEP, pH 7.0). The purified proteins obtained were immediately subjected to fluorescence labeling.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Correlation between Cd2+ binding and transport. A, ITC analysis of Cd2+ binding. Upper panel shows calorimetric titrations of 0.5 mM CdCl2 into 0.01 mM YiiP, YiiPD157A, and YiiPD157C as indicated. Lower panel displays the integrated heats from the upper panel as a function of the Cd2+/protein molar ratio. Solid lines represent best fits of the binding isotherms with fitting parameters summarized in TABLE ONE. B, kinetics of Cd2+ transport. YiiP and mutants were reconstituted into lipids at a molar ratio of 1:20,000 (protein/lipid). Stopped-flow traces of YiiP, YiiPD157A, and YiiPD157C as indicated were generated by 1:1 mixing of proteoliposomes with a series of transport assay buffers containing 0, 0.25, 0.5, 1, 2, or 4 mM CdSO4. Inset, plot of 1/Kobs as a function of 1/[M]. The solid line represents linear regression of the kinetic data.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Correlation between Cd²⁺ Binding and Transport—The transport of metal ions is a sequential process of equilibrium binding and energized movement of metal ions along one or more binding sites in a translocation pathway across the membrane (20). To establish the functional role of Asp-157 in metal binding and transport, we examined the effects of mutating Asp-157 to a cysteine or alanine residue, corresponding to a metal coordination or noncoordination residue. His-tagged YiiP and mutants were overexpressed and purified to homogeneity by nickel affinity chromatography, followed by thrombin cleavage of the affinity tag, EDTA chelation of bound metal ions, and size exclusion HPLC purification to yield protein samples suitable for metal calorimetric titrations (15). Cd²⁺ binding to YiiP, YiiP_D157A, and YiiP_D157C was examined directly by ITC at 25 °C, pH 7.0, as described under "Experimental Procedures." Examples of heat changes resulting from binding of incremental additions of Cd²⁺ and plots of the integrated heat per mol of Cd²⁺ as a function of the Cd²⁺/protein molar ratio are displayed in Fig. 1A. The heat effects generated by Cd²⁺ binding to YiiP and YiiP_D157C dropped sharply near a stoichiometric equivalence point of 2.5, whereas the midpoint of binding heat changes for YiiP_D157A occurred at 1.5, indicating a loss of 1 eq Cd²⁺-binding site by the D157A mutation. Accordingly, binding isotherms were fitted with a two-site model for YiiP and YiiP_D157C and a one-site model for YiiP_D157A. As shown in TABLE ONE, binding affinities, stoichiometries, and H changes of site 1 and site 2 for YiiP and YiiP_D157C are nearly identical and within experimental errors. Fit of YiiP_D157A binding isotherm to a one-site model resulted in K_a = 7.3 ± 1.9 µM^–1, n = 1.5 ± 0.1, and H =–5.7 ± 0.2 kcal/mol, in excellent agreement with the binding parameters of site 1 of both YiiP and YiiP_D157C. Thus, a D157C mutation caused no change to both sites 1 and 2, whereas a D157A mutation completely abolished Cd²⁺ binding to site 2 but had no effect on site 1. Furthermore, the binding stoichiometries for site 2 were close to unity, suggesting that a D157A mutation disrupted one Cd²⁺-binding site per YiiP subunit.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. Correlation between Zn2+ binding and transport. A, ITC analysis of Zn2+ binding. Titrations were made with 5-µl injections of 0.5 mM ZnCl2 into 0.01 mM YiiP, YiiPD157A, and YiiPD157C as indicated. Negative or positive heat effects correspond to an exothermic or endothermic reaction, respectively. Solid lines represent best fits of Zn2+ binding isotherms to a two-site model with fitting parameters summarized in TABLE ONE. B, kinetics of Zn2+ transport. YiiP and mutants were reconstituted into lipids at a molar ratio of 1:20,000 (protein/lipid). Stopped-flow traces of YiiP, YiiPD157A, and YiiPD157C as indicated were fluorescence responses to 0, 0.25, 0.5, 1, 2, or 4 mM ZnSO4 in a transport assay buffer. Inset, plot of 1/Kobs as a function of 1/[M]. The solid line represents linear regression of the kinetic data.

Correlation between Zn²⁺ Binding and Transport—In contrast to the pure exothermic heat reactions during Cd²⁺ titrations, Zn²⁺ titrations shown in Fig. 2A displayed a mixed heat reaction that began with exothermic and followed by late endothermic heat changes. In addition to the main exothermic-to-endothermic transition, another enthalpic transition was evident at the beginning of Zn²⁺ titrations where exothermic heat effects increased progressively. The overall profile of the YiiP_D157C binding isotherm was comparable with that of YiiP, but the binding isotherm of YiiP_D157A showed a leftward shift of the exothermic-to-endothermic transition by about 1 stoichiometry unit, qualitatively corresponding to the loss of one exothermic binding site. To a first approximation, Zn²⁺ binding isotherms were fitted with two sets of independent binding sites, accounting for the endothermic and exothermic heat reactions with the respective binding stoichiometries of 0.6 ± 2.4 and 2.0 ± 0.1 for YiiP, 0.6 ± 1.8 and 1.9 ± 0.1 for YiiP_D157C, and 0.7 ± 3.1 and 1.1 ± 0.1 for YiiP_D157A (TABLE ONE). Compared with YiiP, YiiP_D157C appeared to retain all Zn²⁺-binding sites, whereas YiiP_D157A was short of one exothermic site. The presence of multiple heat transitions precluded fitting of Zn²⁺ binding isotherms with certainty, as indicated by significant fitting errors. Thus a quantitative comparison of Zn²⁺ binding parameters was not amenable. Nevertheless, it was evident that a Zn²⁺ exothermic binding site was disrupted in YiiP_D157A, whereas the same site remained intact in YiiP_D157C. Furthermore, multiple transitions observed in the YiiP_D157A binding isotherm indicated the presence of at least two additional Zn²⁺-binding sites after disruption of the Asp-157 site. In corroboration of the effects of Asp-157 mutations on Zn²⁺ binding, the rate of Zn²⁺ transport was unchanged with k₃ values of 34 ± 5 and 33 ± 3 s^–1 for YiiP and YiiP_D157C, respectively, making a sharp contrast to YiiP_D157A responses that were reduced to the background level (Fig. 2B). The K_m values estimated from three experiments were 310 ± 32 and 358 ± 36 µM for YiiP and YiiP_D157C, respectively, indicating no significant change in Zn²⁺ binding.

Topological Mapping of Asp-157—A mechanistic understanding of the functional role of Asp-157 depends heavily on the ability of mapping it to a reliable topology model. The membrane topology of any CDF transporter has not yet been determined experimentally, but six segments of hydrophobic residues are suggested by YiiP hydropathy analysis. To determine whether each of these hydrophobic segments actually traversed the membrane and, if so, to determine their membrane spanning polarity, we introduced a series of cysteine substitution mutations, each located at the beginning or end of a hydrophobic segment. When intact cells were exposed to an impermeant thiol-specific probe, the probe accessibility to a reporter cysteine residue could indicate its extracellular or intracellular location. Two maleimide derivatives, FM and MPB, were used in this study. The maleimide group in FM is directly attached to a bulky and charged fluorescein moiety, and the maleimide in MPB is tethered to its biotin moiety through a linear polyethylene oxide chain (Fig. 3A). The bulkiness and the hydrophilic nature of both maleimide derivatives make them impermeant to the cytoplasmic membrane (21). YiiP contains two native cysteine residues at positions 127 and 287. FM or MPB labeling to Cys-127 was not detected when YiiP_C287S was exposed to 1 mM FM or MPB, even in the presence of 10% SDS (Fig. 3B). Thus a panel of single cysteine substitution mutants was constructed on the YiiP_C287S background. Only one reactive cysteine residue is present in each of the following YiiP variants at a position as numbered: S10C/C287S, S36C/C287S, N70C/C287S, S107C/C287S, S144C/C287S, R177C/C287S, Cys-287 (equivalent to wild type YiiP). All these mutants were found to be fully functional by stopped-flow analysis, thereby ascertaining the structural and functional relevance of the topological mapping.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Topology analysis. A, molecule structures of FM and MPB. B, YiiPC287S (containing a cysteine residue at position 127) is chemically silent to FM and MPB labeling. MPB labeling was performed with intact cells (lane 1) or sonicated cells (lane 2) as described under "Experimental Procedures." As a control, FM labeling was performed with purified YiiPC287S without MPB pretreatment (lane 3). MPB-treated YiiPC287S was purified and then labeled with FM in the presence of 10% SDS. FM and MPB labeling were detected by UV transillumination (middle) and Western blot (bottom). Approximately an equal amount of protein (3 µg) was loaded to each lane as indicated by Coomassie Blue staining (top), but neither fluorescence transillumination nor Western blot detection yielded any visible signal. C, alternating patterns of FM and MPB labeling. MPB labeling was carried out in intact cells (lane 1) and sonicated cells (lane 2). Lane 3 is FM labeling to purified proteins without MPB pretreatment. Based on the accessibility of these seven cysteine residues to MPB and FM labeling, a topology model is depicted with both N and C terminus in the cytoplasm.

View larger version (7K): [in this window] [in a new window]   FIGURE 4. Probing the hydrophobic core of TM-5. A, positional dependence of FM reactivity. FM labeling was performed with sonicated cells. After FM labeling, seven YiiP variants as indicated were purified, subjected to SDS-PAGE, and then visualized using a UV transilluminator (bottom). Aliquots of purified proteins were exposed to 0.1 mM fresh FM for a secondary FM labeling in the presence of 10% SDS (middle). After fluorescence detection, the same gel was stained with Coomassie Blue (top). B, helical mode of TM-5 with a hydrophobic core from Gln-155 to Ser-171 as indicated. C, Western blot analysis of MPB labeling to six positions as indicated in TM-5. Intact (I) or sonicated cells (S) were used for MPB labeling.

View larger version (8K): [in this window] [in a new window]   FIGURE 5. Protection of MPB labeling to D157C by metal binding. A, specificity of metal protection. MPB labeling was carried out in sonicated cells in the presence of one of the nine divalent metal ions (0.2 mM) or EDTA (0.2 mM) as indicated. Reactions were quenched by 20 mM -ME, and then proteins were purified and applied to SDS-PAGE. MPB labeling was visualized by Western blot (lower panel), and a duplicated gel was stained with Coomassie Blue (upper panel). B, concentration dependence of Zn2+ protection. MPB labeling was carried out with sonicated cells in the presence of Zn2+ at an indicated concentration. C, concentration dependence of Cd2+ protection. MPB labeling was carried out in the presence of Cd2+ at an indicated concentration.

Selective Metal Binding to D157C—Because YiiP_D157C exhibited intact Zn²⁺/Cd²⁺ binding and full Zn²⁺/Cd²⁺ transport activity, the selectivity and binding affinity of metal binding to Cys-157 may reflect metal binding to the native Asp-157. Metal binding to Cys-157 was indicated by metal protection of Cys-157 from MPB-labeling, because metal binding to a membrane-embedded Cys-157 is expected to incur steric hindrance to MPB labeling. YiiP_D157C/C287S in the membrane was exposed to MPB labeling with a brief sonication in the presence of one of the following divalent cations: Zn²⁺, Cd²⁺, Hg²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Ni²⁺ and Co²⁺, each at a concentration of 0.2 mM. The MPB-treated YiiP_D157C/C287S was then purified and subjected to Western blot analysis. Among the nine divalent metals tested, only Zn²⁺ and Cd²⁺ appeared to abolish the corresponding Western blot signals (Fig. 5A, lower panel), whereas Coomassie Blue staining of a duplicate gel indicated that approximately an equal amount of protein was loaded to each lane (Fig. 5A, upper panel). Additional experiments were carried out to examine the concentration dependence of Zn²⁺ or Cd²⁺ protection. MPB labeling was carried out with sonicated cells in the presence of Zn²⁺ or Cd²⁺ with a concentration ranging from 0 to 128 µM as indicated. The intensity of MPB labeling began to decrease at 16 µM for Zn²⁺ (Fig. 5B) and 32 µM for Cd²⁺ (Fig. 5C), and then rapidly approached a background level at 64 µM, indicating that the equilibrium binding of both metal ions could effectively protect the irreversible MPB labeling at 32–64 µM, at which Zn²⁺/Cd²⁺ binding to Cys-157 must have approached saturation. Thus, the binding affinity of Zn²⁺/Cd²⁺ to Cys-157 might be in a micromolar range.

View larger version (9K): [in this window] [in a new window]   FIGURE 6. ITC analyses of Fe2+ and Hg2+ binding. A, titrations with 5-µl injections of 0.25 mM FeCl2 into 0.01 mM YiiP or YiiPD157A as indicated. B, titrations with 5-µl injections of 0.5 mM HgCl2 into 0.01 mM YiiP or YiiPD157A as indicated. The solid lines represent the best fits to a two-site binding model with fitting parameters summarized in TABLE TWO.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The correlation between Zn²⁺/Cd²⁺ binding and transport distinguishes Asp-157 from a regular metal coordination residue. Besides supplying an oxygen donor for coordination binding, Asp-157 also acts as a pivotal structural component that couples metal binding to a transport process. Thus, the local chemical environment surrounding Asp-157 may contribute to binding-transport coupling. Asp-157 was localized to TM-5 by coarse topological mapping using a set of cysteine substitution mutations introduced such that each pair of cysteine residues was positioned in two solvent-accessible regions flanking each of the six putative TMs in YiiP. Thiol-specific labeling using impermeant probes MPB and FM revealed an alternating pattern of thiol reactivities when intact cells were probed, indicating that the polypeptide chain of YiiP traverses the membrane six times with both N and C termini in the cytoplasm. A fine topological mapping then focused on TM-5 by using cysteine-scanning mutagenesis to probe the positional dependence of cysteine thiol reactivities toward a water-soluble probe, FM, which reacts more readily with solvent-exposed cysteines than with those embedded in the membrane. A central region of low and two peripheral regions of higher FM reactivities were identified with two sharp boundaries occurring between residues 153–155 and 171–172. The lower FM reactivities could be attributed to inaccessibility (23) and/or elevated pK_a of cysteine residues (24) in a hydrophobic environment. Thus Asp-157 was localized to a stretch of membrane-embedded residues. The mechanistic implications of a membrane-embedded Asp-157 are 2-fold. 1) The transport of metal ions is thought to be associated with a global protein conformational change originating from the metal-binding site. The hydrophobic surroundings of Asp-157 can greatly strengthen the Asp-157-Zn²⁺/Cd²⁺ interaction and facilitate electrostatic propagation of a local conformational change through a hydrophobic medium. 2) Zn²⁺ transport in YiiP is coupled to antiport of proton(s). The localization of Asp-157 to a hydrophobic environment raises the possibility that the pK_a of this membrane-embedded aspartate may be sufficiently elevated to remain protonated in the absence of Zn²⁺/Cd²⁺ binding. Upon Zn²⁺/Cd²⁺ binding, Asp-157 may be deprotonated to trigger Zn²⁺/Cd²⁺ transport with a separation of the released proton moving in an opposite direction. In agreement with this speculation, our recent ITC study indicated that binding one Cd²⁺ to YiiP yielded 1.23 protons (15). Further experiments are underway to investigate the involvement of Asp-157 in binding-deprotonation coupling.

CDF transporters were initially identified as Zn²⁺/Cd²⁺/Co²⁺ pumps (25,26), and they subsequently were shown to transport Cd²⁺, Mn²⁺, Ni²⁺, and Fe²⁺. Categorization of CDF proteins based on multiple sequence alignment suggested three distinct subgroups, each apparently associated with a broad range of substrate specificities but with different metal preferences (3). The E. coli CDF transporters ZitB and YiiP belong to group 2 and group 3, respectively. ZitB functions as a Zn²⁺/Cd²⁺ efflux pump, whereas the substrate specificity of YiiP remains obscure. YiiP (FieF) was recently implicated in a role of iron efflux pump based on the observations that both zinc and iron induced yiiP transcription and that overexpression of YiiP led to a loss of cytosolic iron content in E. coli and an increase of iron tolerance in a fur strain. However, only accumulation of Zn²⁺ but not Fe²⁺ in everted membrane vesicles was established, although Fe²⁺ transport was observed in reconstituted YiiP proteoliposomes (12). In the present study, both MPB protection analysis and direct ITC measurement indicated that Fe²⁺ did not bind to Asp-157 that was localized to the active site of the Zn²⁺/Cd²⁺ transporter. Nevertheless, Fe²⁺ calorimetric titrations suggested the presence of two independent Fe²⁺-binding sites in YiiP, both with modest binding affinities (2.2 and 0.22 µM). The functional connection of these putative Fe²⁺-binding sites to Fe²⁺ transport has yet to be established.

Contrary to the general expectation that YiiP is a broad range metal ion transporter, metal binding to Asp-157 is highly specific. Calorimetric measurements of metal binding to YiiP_Asp-157 and YiiP_D157A showed that a D157A mutation disrupted Zn²⁺ or Cd²⁺ binding to Asp-157 but detectable caused no change to Fe²⁺ or Hg²⁺ binding. Fe²⁺ and Hg²⁺ represent neighboring metal ions in the same transition period and in the same element group, respectively. The selectivity of Asp-157 for Zn^2+/Cd²⁺ over Fe²⁺ and Hg²⁺ is consistent with the selectivity of Cys-157 for Zn²⁺/Cd²⁺ over Fe²⁺ and Hg²⁺, as well as Co²⁺, Ni²⁺, Mn²⁺, Ca²⁺, and Mg²⁺, as suggested by metal binding protection of Cys-157 from MPB labeling. The lack of Hg²⁺ protection is noteworthy because the cysteine thiolate is a very strong Hg²⁺ donor group. It is difficult to see how selection of Zn²⁺/Cd²⁺ by Asp-157 and Cys-157 is so specifically based on chemical consideration for metal ions, because the electrostatic binding by Zn²⁺/Cd²⁺ is a property shared with Mg²⁺ and Ca ⁺, whereas the Lewis acid strength, and thus the donor group preference of Zn²⁺/Cd²⁺, is within the same range shared by Co²⁺, Ni²⁺, Fe²⁺, and Mn²⁺. The mechanism of metal selectivity may be better appreciated in terms of coordination chemistry in the chemical context of the immediate binding site neighborhood. To a first approximation, the hydrophobic surroundings of Asp-157 may entail steric and electrostatic hindrances as suggested by the observation that Cys-157 was only partially accessible to thiol-specific modification by a maleimide group tethered to a flexible and neutral MPB as opposed to a rigid and charged FM molecule.

Previous ITC analyses of metal binding to YiiP suggested the presence of at least one mutually competitive binding site common to Zn²⁺, Cd²⁺, and Hg²⁺ and a set of noncompetitive binding sites (15). In the present study, we physically assigned Asp-157 to one of the noncompetitive to binding sites, because metal binding to Asp-157 is specific to Zn²⁺ and Cd²⁺ but not to Hg²⁺. After removing the Asp-157 site by a D157A mutation, a reduced Cd ⁺ binding isotherm could be fitted with one set of independent binding sites with a binding stoichiometry of 1.5, suggesting that one or two additional Cd²⁺-binding sites are present in YiiP_D157A. In light of our recent finding that YiiP is a homodimer (27), the 1.5 stoichiometric equivalence of Cd²⁺ binding may be interpreted as two independent Cd²⁺-binding sites, one is located in each subunit and the other at the dimeric subunit interface. The molecular identities of these additional Cd²⁺-binding sites and their roles in metal transport have yet to be determined. The stoichiometries of Zn²⁺ and Cd²⁺ binding to Asp-157 are both close to unity, corresponding to two independent Asp-157-binding sites per YiiP homodimer. Because Zn²⁺/Cd²⁺ binding to Asp-157 is directly linked to Zn²⁺/Cd²⁺ transport, transport Zn²⁺/Cd²⁺ in a YiiP dimer may occur in two independent translocation pathways, each located in the center of a YiiP monomer. Alternatively, two Cd -binding sites may be located in a common translocation pathway at the dimer interface, thus transport of two Cd²⁺ ions may occur in a shared translocation pathway. Further structural analysis of YiiP is underway to test these hypotheses.

	FOOTNOTES

 
^* This work was supported by the Laboratory Directed Research and Development Program of the Brookhaven National Laboratory, Project 05-064 (to Y. W. and D. F.), and by a National Institutes of Health Grant RO1 GM65137 (to D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Goldhabor postdoctoral fellow.

² To whom correspondence should be addressed: Biology Dept., Bldg. 463, Brookhaven National Laboratory, Upton, NY 11973. Tel.: 631-344-4208; Fax: 631-344-3407: E-mail: dax{at}bnl.gov.

³ The abbreviations used are: CDF, cation diffusion facilitator; -ME, -mercaptoethanol; DDM, n-dodecyl--D-maltoside; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride; ITC, isothermal titration calorimetry; FM, fluorescein 5-maleimide; MPB, maleimide polyethylene oxide (PEO)₂ biotin; HPLC, high pressure liquid chromatography.

	ACKNOWLEDGMENTS

 
We thank John J. Dunn for providing sequencing service. BNL is managed by Brookhaven Science Associates for the United States Department of Energy.

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