Selective Metal Binding to a Membrane-embedded Aspartate in the Escherichia coli Metal Transporter YiiP (FieF)
2005; Elsevier BV; Volume: 280; Issue: 40 Linguagem: Inglês
10.1074/jbc.m506107200
ISSN1083-351X
Autores Tópico(s)Extraction and Separation Processes
ResumoThe 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 YiiPD157A 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. 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 YiiPD157A 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. Membrane transporters in the cation diffusion facilitator family are found both in eukaryotes and prokaryotes (1Paulsen I. Saier M.J. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (295) Google Scholar). 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 (2Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: 138-141Crossref PubMed Google Scholar). Despite sequence variability, all CDF 3The abbreviations used are: CDF, cation diffusion facilitator; β-ME, β-mercaptoethanol; DDM, n-dodecyl-β-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. 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 (3Nies D.H. FEMS Microbiol. Rev. 2003; 27: 313-339Crossref PubMed Scopus (1092) Google Scholar, 4Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (429) Google Scholar, 5Hantke K. Biometals. 2001; 14: 239-249Crossref PubMed Scopus (150) Google Scholar, 6Harris E. Nutr. Rev. 2002; 60: 121-124Crossref PubMed Scopus (45) Google Scholar, 7Haney C.J. Grass G. Franke S. Rensing C. J. Ind. Microbiol. Biotechnol. 2005; 12: 213-226Google Scholar). 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+ (8Anton A. Grosse C. Reissmann J. Pribyl T. Nies D.H. J. Bacteriol. 1999; 181: 6876-6881Crossref PubMed Google Scholar, 9Persans M.W. Nieman K. Salt D.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9995-10000Crossref PubMed Scopus (261) Google Scholar, 10Bloss T. Clemens S. Nies D.H. Planta (Heidelberg). 2002; 214: 783-791Crossref PubMed Scopus (87) Google Scholar, 11Delhaize E. Kataoka T. Hebb D.M. White R.G. Ryan P.R. Plant Cell. 2003; 15: 1131-1142Crossref PubMed Scopus (210) Google Scholar, 12Grass G. Otto M. Fricke B. Haney C.J. Rensing C. Nies D.H. Munkelt D. Arch. Microbiol. 2005; 183: 9-18Crossref PubMed Scopus (163) Google Scholar, 13Munkelt D. Grass G. Nies D.H. J. Bacteriol. 2004; 186: 8036-8043Crossref PubMed Scopus (85) Google Scholar). 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 (12Grass G. Otto M. Fricke B. Haney C.J. Rensing C. Nies D.H. Munkelt D. Arch. Microbiol. 2005; 183: 9-18Crossref PubMed Scopus (163) Google Scholar, 14Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). 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 (14Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). 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 (15Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). 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 (12Grass G. Otto M. Fricke B. Haney C.J. Rensing C. Nies D.H. Munkelt D. Arch. Microbiol. 2005; 183: 9-18Crossref PubMed Scopus (163) Google Scholar). 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 (16Frausto da Silva J. Williams R. The Biological Chemistry of the Elements: The Inorganic Chemistry of Life. Oxford University Press, Oxford, UK2001: 39-51Google Scholar). 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 (17Anton A. Weltrowski K. Klebba A. Haney C. Grass G. Rensing C. Dietrich H. J. Bacteriol. 2004; 186: 7499-7507Crossref PubMed Scopus (102) Google Scholar). The Asp appeared to be essential because expression of CzcDD158E, CzcDD158A, ZitBD163E, and ZitBD163A 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. Site-directed Mutagenesis—Cloning and construction of the expression plasmid pYiiP-His were described previously (15Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). 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 (18Studier F.W. Protein Expression Purif. 2005; 41: 207-234Crossref PubMed Scopus (4140) Google Scholar). Cells from overnight cultures were harvested, and membrane proteins were extracted using 7% n-dodecyl-β-d-maltopyranoside (DDM) as described previously (15Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). 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 3000SWXL 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 (15Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). 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 ΔH, association constant Ka 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, YiiPD157C, or YiiPD157A was reconstituted into liposomes made of E. coli polar lipids (Avanti Polar Lipids) as described previously (14Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). 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 ΔF. ΔF/ΔFmax was obtained by normalizing ΔF to the maximum proteoliposome response elicited by a transport assay buffer containing 4 mm ZnSO4 or CdSO4 plus 2% n-octyl-β-d-glucoside used to solubilized proteoliposomes. Determination of Transport Kinetic Parameters—The rate of the fluorescence rise kobs 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 (14Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), M+T1↔k1/k2MT1→k3T2+MSCHEME 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 Kd = k2/k1. The relationship among k1, k2, and k3 is defined as Km = (k2 + k3)/k1. Application of the steady-state condition to the species MT1 gives Equation 1 as described previously (19Strickland S. Palmer G. Massey V. J. Biol. Chem. 1975; 250: 4048-4052Abstract Full Text PDF PubMed Google Scholar). Kobs=k1k3[M]k1[M]+k2+k3(Eq. 1) Thus, Equation 2, 1Kobs=1k3+Kmk3[M](Eq. 2) k3 and Km were determined by linear regression of 1/Kobs 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 β-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 × 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. Fluorescence Labeling and Western Blot—The MPB-treated and purified proteins (∼0.5 mg/ml) were incubated with 0.1 mm fluorescein 5-maleimide (FM, Molecular Probes) in the presence of 10% SDS at room temperature for 20 min, and then β-ME was added to 10 mm to terminate the reaction. The resulting FM-treated proteins were subjected to SDS-PAGE on an 8–16% Tris-HCl precast polyacrylamide gel (Bio-Rad). The gel was visualized on a UV transilluminator and documented using a BioDoc-It System (Ultraviolet Products). After fluorescence detection, proteins were transferred to nitrocellulose using a Trans-blot semi-dry transfer cell (Bio-Rad) and were exposed to a peroxidase-conjugated monoclonal anti-biotin antibody (Sigma) for detection of MPB labeling by a SuperSignal West Pico chemiluminescent substrate (Pierce). For FM labeling to membrane-bound YiiP variants, cells hosting the overexpression of a YiiP variant were harvested and resuspended in the reaction buffer, and then 1 mm FM was added with a brief sonication. After incubation for 30 min at room temperature, 10 mm β-ME was added to quench unreacted FM. The resulting cells were pelleted and washed, and the FM-treated proteins were purified as described above. Aliquots of purified proteins were either directly subjected to SDS-PAGE or subjected to another exposure of 0.1 mm fresh FM in the presence of 10% SDS, followed by SDS-PAGE. Proteins in the gels were visualized under UV light for fluorescence detection before being stained with Coomassie Blue for estimation of the total amount of proteins. 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 (20Jardetzky O. Nature. 1966; 211: 969-970Crossref PubMed Scopus (884) Google Scholar). 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 (15Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Cd2+ binding to YiiP, YiiPD157A, and YiiPD157C 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 YiiPD157C dropped sharply near a stoichiometric equivalence point of 2.5, whereas the midpoint of binding heat changes for YiiPD157A 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 YiiPD157C and a one-site model for YiiPD157A. As shown in TABLE ONE, binding affinities, stoichiometries, and ΔH changes of site 1 and site 2 for YiiP and YiiPD157C are nearly identical and within experimental errors. Fit of YiiPD157A binding isotherm to a one-site model resulted in Ka = 7.3 ± 1.9 μm–1, n = 1.5 ± 0.1, and ΔH =–5.7 ± 0.2 kcal/mol, in excellent agreement with the binding parameters of site 1 of both YiiP and YiiPD157C. 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.TABLE ONESummary of Cd2+ and Zn2+ binding parametersTitrantProteinSite 1Site 2nKaΔHnKaΔHμm-1kcal/molμm-1kcal/molCdCl2YiiP1.5 ± 0.17.9 ± 2.7-5.8 ± 0.21.1 ± 0.11.1 ± 0.5-7.8 ± 0.7YiiPD157A1.5 ± 0.17.3 ± 1.9-5.7 ± 0.2YiiPD157C1.4 ± 0.17.5 ± 1.8-5.5 ± 0.11.2 ± 0.11.1 ± 0.3-6.9 ± 0.3ZnCl2YiiP0.6 ± 2.40.0069 ± 0.002922 ± 862.0 ± 0.14.2 ± 14-3.1 ± 0.2YiiPD157A0.7 ± 3.10.061 ± 0.0113.4 ± 1.71.1 ± 0.126 ± 14-2.7 ± 0.2YiiPD157C0.6 ± 1.80.0079 ± 0.002135 ± 111.9 ± 0.114 ± 29-1.7 ± 0.9 Open table in a new tab The ITC data indicated the presence of at least two Cd2+-binding sites, each of which could play a structural, functional, or regulatory role. To examine a possible correlation between Cd2+ binding to Asp-157 and Cd2 transport, HPLC-purified YiiP, YiiPD157A, and YiiPD157C were reconstituted into proteoliposomes, and the kinetics of Cd2+ transport was analyzed by stopped-flow measurements of fluorescence changes of an encapsulated Zn2+/Cd2+-sensitive indicator, fluozin-1, in response to rapid mixtures of proteoliposomes with Cd2+ exterior to vesicles. SDS-PAGE analysis of proteoliposome samples confirmed that approximately the same amount of YiiP, YiiPD157A, and YiiPD157C was reconstituted into vesicles. Mixing proteoliposomes with external Cd2+ evoked rapid and progressive fluorescence increases that were dependent of Cd2+ concentrations ranging from 0 to 2 mm in the reaction mixture as described under "Experimental Procedures." Liposomes prepared in parallel to proteoliposomes only yielded negligible background fluorescence responses. A linear correlation between the initial rates of fluorescence responses and the molar ratios of YiiP/lipid was observed, indicating a linear relationship between the initial rate and the transport activity (data not shown). As shown in Fig. 1B, the fluorescence responses of YiiP and YiiPD157C were comparable in amplitudes within the Cd2+ concentration range, in contrast to greatly diminished responses of YiiPD157A that were reduced to a background level. This significant impairment of the transport activity was attributed to the removal of a metal coordination group by a D157A mutation, because an Asp → Cys substitution only caused insignificant kinetic changes within experimental errors. Linear regression of 1/kobs versus 1/[Cd2+] using kinetic data from three experiments (Fig. 1B, inset) yielded Km values of 266 ± 20 and 304 ± 32 μm for YiiP and YiiPD157C, respectively. The k3 values for YiiP and YiiPD157C were identical and within experimental errors (26 ± 6 s–1). 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 YiiPD157C binding isotherm was comparable with that of YiiP, but the binding isotherm of YiiPD157A 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 YiiPD157C, and 0.7 ± 3.1 and 1.1 ± 0.1 for YiiPD157A (TABLE ONE). Compared with YiiP, YiiPD157C appeared to retain all Zn2+-binding sites, whereas YiiPD157A 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 YiiPD157A, whereas the same site remained intact in YiiPD157C. Furthermore, multiple transitions observed in the YiiPD157A 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 YiiPD157C, respectively, making a sharp contrast to YiiPD157A responses that were reduced to the background level (Fig. 2B). The Km values estimated from three experiments were 310 ± 32 and 358 ± 36 μm for YiiP and YiiPD157C, 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 (21Fann M.C. Maloney P.C. J. Biol. Chem. 1998; 273: 33735-33740Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). YiiP contains two native cysteine residues at positions 127 and 287. FM or MPB labeling to Cys-127 was not detected when YiiPC287S 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 YiiPC287S 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. MPB labeling was carried out with intact cells, from which MPB-treated YiiP variants were purified, and then exposed to FM in 10% SDS. The resultant MPB-FM doubly treated YiiP variants were separated on a SDS-polyacrylamide gel for transillumination of FM labeling and then Western blot detection of MPB labeling. As shown in Fig. 3C, Western blots revealed a strong protein band in lane 1 for S36C, S107C, and R177C (top panel) but no detectable signal for S10C, N70C, S144C, and Cys-287 (bottom panel). The MPB reactivities in the former three positions and the lack of MPB reactivity in the latter four positions mirrored the pattern of FM fluorescence labeling, showing a background level of fluorescence in Fig. 3C, lane 1, for S36C, S107C, and R177C, as opposed to an intense fluorescence signal in lane 1 for S10C, N70C, S144C, and Cys-287. Because MPB labeling was directed to cysteine residues on the extracellular surface, whereas FM labeling was directed to any cysteine residue that su
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