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• W2012087369 abstract "For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of ∼300 nm. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore. For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of ∼300 nm. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore. Iron, an essential nutrient for almost all bacterial species, is required for metabolic processes, including electron transfer, oxygen activation, and biosynthesis of amino acids and nucleosides (1Wandersman C. Delepelaire P. Annu. Rev. Microbiol. 2004; 58: 611-647Crossref PubMed Scopus (724) Google Scholar). However, Fe3+ is scarce in the extracellular environment. Gram-negative bacteria have evolved transport processes that utilize siderophores to scavenge extracellular Fe3+ by high affinity chelation. Different siderophore receptors are expressed at the bacterial outer membrane (OM), 8The abbreviations used are: OM, outer membrane; CM, cytoplasmic membrane; Fcn, ferricrocin; DLS, dynamic light scattering; SPR, surface plasmon resonance; r.m.s.d., root mean square deviation; BSA, bovine serum albumin; MBP, maltose-binding protein; AEDANS, 5-((((2-iodoacetyl)amino)-ethyl)amino)naphthaline-1-sulfonic acid; RU, response units; MDCC, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin; PDB, Protein Data Bank; DNS, discrete noninteracting species; Rh, hydrodynamic radius.8The abbreviations used are: OM, outer membrane; CM, cytoplasmic membrane; Fcn, ferricrocin; DLS, dynamic light scattering; SPR, surface plasmon resonance; r.m.s.d., root mean square deviation; BSA, bovine serum albumin; MBP, maltose-binding protein; AEDANS, 5-((((2-iodoacetyl)amino)-ethyl)amino)naphthaline-1-sulfonic acid; RU, response units; MDCC, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin; PDB, Protein Data Bank; DNS, discrete noninteracting species; Rh, hydrodynamic radius. each with specificity for a particular metal-chelated siderophore. Transport of receptor-bound siderophores into the periplasm requires contribution of energy provided by the TonB-ExbB-ExbD complex that is anchored in the cytoplasmic membrane (CM). TonB spans the periplasm to make contacts with cognate OM receptors. By harnessing energy produced from the proton motive force, TonB may propagate conformational changes to OM siderophore receptors, resulting in release of siderophore into the periplasm. The ferrichrome transport system consists of four proteins (FhuA, FhuB, FhuC, and FhuD) expressed by Gram-negative bacteria. The FhuA protein consists of two domains as follows: an N-terminal globular cork domain is enclosed by a 22-stranded C-terminal β-barrel domain (2Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (657) Google Scholar, 3Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosen-busch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Connections between β-strands in the barrel domain are such that long loops participating in ferrichrome binding are exposed to the extracellular environment; short turns are exposed to the periplasm. Uptake of ferrichrome is a TonB-dependent process, mediated by contacts between TonB and the OM receptor FhuA. TonB is elongated and has three domains as follows: an N-terminal domain anchored in the CM, an intermediate domain containing Pro-Glu and Pro-Lys repeats, and a globular C-terminal domain with a central β-sheet and two α-helices. To date, structural data are only available for the C-terminal domain (4Chang C. Mooser A. Plückthun A. Wlodawer A. J. Biol. Chem. 2001; 276: 27535-27540Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 5Ködding J. Killig F. Polzer P. Howard S.P. Diederichs K. Welte W. J. Biol. Chem. 2005; 280: 3022-3028Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Peacock R.S. Andrushchenko V.V. Demcoe A.R. Gehmlich M. Lu L.S. Herrero A.G. Vogel H.J. Biometals. 2006; 19: 127-142Crossref PubMed Scopus (14) Google Scholar). We recently solved the crystal structure of the 1:1 TonB-FhuA complex (7Pawelek P.D. Croteau N. Ng-Thow-Hing C. Khursigara C.M. Moi-seeva N. Allaire M. Coulton J.W. Science. 2006; 312: 1399-1402Crossref PubMed Scopus (193) Google Scholar). The C-terminal domain of TonB makes extensive contacts with the N-terminal consensus Ton box of FhuA, as well as residues Ala-26 and Glu-56 of the cork domain and with periplasmic turns 8 and 10. These contacts orient TonB such that it may mediate conformational disruption of the internal cork domain of FhuA, allowing for passage of siderophore into the periplasm. Although recent structural and biophysical data have clarified initial steps of the siderophore transport cycle involving TonB-receptor interactions, little is known about the fate of the siderophore once transported into the periplasm. Specifically, the molecular mechanisms of siderophore transport from periplasm to cytoplasm are largely uncharacterized. FhuD in the periplasm binds the hydroxamate siderophores ferrichrome, coprogen, and aerobactin (8Köster W. Braun V. J. Biol. Chem. 1990; 265: 21407-21410Abstract Full Text PDF PubMed Google Scholar). Loss of FhuD function in vivo prevented growth of Escherichia coli under iron-limiting conditions when ferrichrome, coprogen, or aerobactin were used as the sole iron source, suggesting that FhuD is a necessary component of the hydroxamate siderophore transport system (8Köster W. Braun V. J. Biol. Chem. 1990; 265: 21407-21410Abstract Full Text PDF PubMed Google Scholar). FhuD was reported to interact with regions of the CM-embedded permease FhuB. Interactions between FhuD and FhuB have been demonstrated by cross-linking studies, protease protection assays (9Rohrbach M.R. Braun V. Köster W. J. Bacteriol. 1995; 177: 7186-7193Crossref PubMed Google Scholar), and enzyme-linked immunosorbent assay (10Mademidis A. Killmann H. Kraas W. Flechsler I. Jung G. Braun V. Mol. Microbiol. 1997; 26: 1109-1123Crossref PubMed Scopus (42) Google Scholar). Interaction of FhuB with FhuD is apparently independent of siderophore binding by FhuD (9Rohrbach M.R. Braun V. Köster W. J. Bacteriol. 1995; 177: 7186-7193Crossref PubMed Google Scholar). Taken together, these results suggest that FhuD functions as a carrier protein; ferrichrome released from the OM receptor is delivered by FhuD to the permease. The integral membrane protein FhuB then translocates the siderophore into the cytoplasm mediated by ATP hydrolysis of FhuC (11Schultz-Hauser G. Köster W. Schwarz H. Braun V. J. Bacteriol. 1992; 174: 2305-2311Crossref PubMed Google Scholar). The crystal structure of FhuD in complex with gallichrome, a ferrichrome analogue, has been reported (12Clarke T.E. Ku S.Y. Dougan D.R. Vogel H.J. Tari L.W. Nat. Struct. Biol. 2000; 7: 287-291Crossref PubMed Scopus (104) Google Scholar), as well as structures of FhuD in complex with albomycin, coprogen, and Desferal® (13Clarke T.E. Braun V. Winkelmann G. Tari L.W. Vogel H.J. J. Biol. Chem. 2002; 277: 13966-13972Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The fold of this 32-kDa protein is bilobal; globular N- and C-terminal domains are connected by a long α-helix that confers rigidity to the protein. The siderophore-binding site residing in a shallow cleft between the two lobes is hydrophobic, having predominantly aromatic residues. Siderophore binds to FhuD through both hydrophobic and hydrophilic interactions. Methylene carbon atoms in the siderophore form hydrophobic interactions with numerous aromatic FhuD residues in the binding cleft. Hydrogen bonds are formed between hydroxamate groups of the siderophore and FhuD residues Arg-84 and Tyr-106. A hydrogen bond with the siderophore is also formed with FhuD residues Asn-215 and Ser-219 through an intermediate water molecule. The overall fold of FhuD is similar to that of BtuF (14Borths E.L. Locher K.P. Lee A.T. Rees D.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16642-16647Crossref PubMed Scopus (176) Google Scholar, 15Karpowich N.K. Huang H.H. Smith P.C. Hunt J.F. J. Biol. Chem. 2003; 278: 8429-8434Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), the periplasmic cobalamin-binding protein of E. coli. Periplasmic metal-binding proteins TroA (16Lee Y.H. Deka R.K. Norgard M.V. Radolf J.D. Hasemann C.A. Nat. Struct. Biol. 1999; 6: 628-633Crossref PubMed Scopus (139) Google Scholar) and PsaA (17Lawrence M.C. Pilling P.A. Epa V.C. Berry A.M. Ogunniyi A.D. Paton J.C. Structure (Lond.). 1998; 6: 1553-1561Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) are also structurally related to FhuD. These proteins share a fold distinct from those of classical periplasmic proteins such as maltose-binding protein (18Krewulak K.D. Peacock R.S. Vogel H.J. Crosa J.H. Mey A.R. Payne S.M. Iron Transport in Bacteria. American Society for Microbiology, Washington, D. C.2004: 113-129Google Scholar). However, unlike maltose-binding protein, FhuD does not exhibit gross conformational rearrangements upon ligand binding. The linker connecting the N-terminal and C-terminal domains in FhuD is a kinked α-helix that crosses these domains only once. The structure of FhuD and the hydrophobicity of the siderophore-binding site suggest that large scale opening and closing of the binding site does not occur upon siderophore binding and release (18Krewulak K.D. Peacock R.S. Vogel H.J. Crosa J.H. Mey A.R. Payne S.M. Iron Transport in Bacteria. American Society for Microbiology, Washington, D. C.2004: 113-129Google Scholar). Molecular dynamics simulations also suggest that FhuD is conformationally rigid but that subtle conformational differences in the C-terminal domain between the apo- and holo-forms may be sufficient for discrimination by FhuB (19Krewulak K.D. Shepherd C.M. Vogel H.J. Biometals. 2005; 18: 375-386Crossref PubMed Scopus (25) Google Scholar). What molecular events result in capture of ferrichrome by FhuD following its TonB-dependent release from FhuA? Given the apparent weak affinity (1 μm) of ferrichrome for FhuD (9Rohrbach M.R. Braun V. Köster W. J. Bacteriol. 1995; 177: 7186-7193Crossref PubMed Google Scholar), binding is unlikely to be a diffusion-governed process. Efficiency of siderophore capture would be enhanced by positioning a binding protein proximal to the lumen of the OM receptor. This organization would promote direct transfer of ferrichrome from FhuA to FhuD. Here we report the first biophysical evidence that TonB specifically interacts with FhuD. Discrete regions of protein-protein interactions on the surfaces of both FhuD and TonB were identified by phage display. Interactions were confirmed by dynamic light scattering, fluorescence spectroscopy, and surface plasmon resonance. Our results suggest that siderophore released from FhuA during the transport cycle is transferred to FhuD via a coordinated transfer mechanism mediated by TonB. Hence, TonB would act as a periplasm-spanning scaffold, directly connecting siderophore transport events between the OM and CM. Bacterial Strains, Phage Libraries, and Media—Random peptide phage libraries Ph.D.-C7C and Ph.D.-12 were purchased from New England Biolabs; E. coli ER2738, also from New England Biolabs, was used for amplification and titration of phage M13 pools. E. coli ER2566 was used to express recombinant TonBs (20Khursigara C.M. De Crescenzo G. Pawelek P.D. Coulton J.W. J. Biol. Chem. 2004; 279: 7405-7412Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar); E. coli BL21 (DE3) pLysS was used to express recombinant FhuDs. Plasmid pCMK01 expresses a hexahistidine-tagged TonB 32-239 (25 kDa; hereafter identified as TonB), and pWA01 expresses a hexahistidine-tagged TonB 32-239 with an engineered cysteine residue at its N terminus (hereafter identified as Cys-TonB) (21Khursigara C.M. De Crescenzo G. Pawelek P.D. Coulton J.W. Biochemistry. 2005; 44: 3441-3453Crossref PubMed Scopus (26) Google Scholar, 22Moeck G.S. Letellier L. J. Bacteriol. 2001; 183: 2755-2764Crossref PubMed Scopus (55) Google Scholar). FhuD was expressed from pMR21 provided by W. Köster (VIDO, Saskatoon, Saskatchewan, Canada); the N terminus of FhuD containing the signal sequence was removed and replaced by a decahistidine tag (23Rohrbach M.R. Paul S. Köster W. Mol. Gen. Genet. 1995; 248: 33-42Crossref PubMed Scopus (13) Google Scholar) (32 kDa; hereafter identified as FhuD). Plasmid pMR21 was commercially mutated to cysteine at Thr-81 by Norclone Biotech Laboratories (London, Ontario, Canada); this protein is hereafter identified as FhuD T181C. Mutagenesis was confirmed by DNA sequencing at Sheldon Biotechnology Centre, McGill University (Montreal, Quebec, Canada). All bacteria were cultured in Luria Bertani (LB) broth containing antibiotics when necessary. Chemicals and Reagents—5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) and isopropyl β-d-thiogalactopyranoside were purchased from BioVectra (Charlottetown, Prince Edward Island, Canada). Protein-grade Tween 20 was purchased from Calbiochem. Antibiotics were purchased from Sigma. Nickel-nitrilotriacetic acid resin used for protein purifications was purchased from Qiagen. The reducing agent tris-(2-carboxyethyl)phosphine and fluorescent dyes 5-((((2-iodoacetyl)amino)-ethyl)amino)naphthaline-1-sulfonic acid (AEDANS) and 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) were purchased from Invitrogen. Protein Purification—TonB and Cys-TonB were purified as described previously (20Khursigara C.M. De Crescenzo G. Pawelek P.D. Coulton J.W. J. Biol. Chem. 2004; 279: 7405-7412Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). To purify overexpressed FhuD or FhuD T181C, cell pellets were suspended in 50 ml of buffer A containing 50 mm Tris, pH 8.2, 150 mm NaCl, and 5 mm imidazole plus one Complete mini EDTA-free protease inhibitor mixture tablet (Roche Applied Science); 0.16 mg/ml lysozyme and 16 μm phenylmethylsulfonyl fluoride were then added. Cells were shaken at room temperature for 30 min, followed by addition of 0.04 mg/ml DNase, 0.04 mg/ml RNase, and an additional 16 μm phenylmethylsulfonyl fluoride. To lyse bacteria, cells were passed twice through an Emulsiflex-C5 (Avestin, Ottawa, Ontario, Canada). Cell lysate was centrifuged (27,000 × g, 4 °C) for 50 min and filtered through 0.45-μm syringe filters. Filtered cell extracts containing FhuD or FhuD T181C were applied to nickel-nitrilotriacetic acid resin equilibrated with buffer A. FhuDs were eluted with 50 mm Tris, pH 8.2, containing 125 mm imidazole, pooled, and applied to a POROS HQ 20 anion exchange column (Applied Biosystems). Bound proteins were washed with 50 mm Tris, pH 8.2, containing 125 mm imidazole, eluted with 160 mm NaCl, and applied inline to a POROS MC 20 column (Applied Biosystems). After extensive washing, proteins were eluted with 50 mm Tris, pH 8.2, and 120 mm imidazole and applied to a second POROS HQ 20 column, washed as described above, and eluted with 180 mm NaCl in 50 mm Tris, pH 8.2. Purified proteins were dialyzed in a 24,000 Mr cut-off dialysis membrane (SpectraPor) for 16 h at 4 °C in 100 mm Hepes, 150 mm NaCl, pH 7.4. Homogeneity of FhuDs was confirmed by SDS-PAGE and silver staining of 750 ng of total protein. Concentrations of protein were determined by either a Bradford or BCA assay using bovine serum albumin as standard. Phage Display—Phage panning against TonB as target was described previously (24Carter D.M. Gagnon J.N. Damlaj M. Mandava S. Makowski L. Rodi D.J. Pawelek P.D. Coulton J.W. J. Mol. Biol. 2006; 357: 236-251Crossref PubMed Scopus (32) Google Scholar). Purified FhuD was diluted to 100 μg/ml in TBS (Tris-buffered saline: 50 mm Tris, pH 7.5, 150 mm NaCl), and 150 μl of protein was adsorbed to a polystyrene microtiter plate (Nunc Maxisorp). Plates coated with immobilized FhuD were incubated for 16 h at 4 °C followed by blocking (2 h at 37 °C) with TBS containing 5 mg/ml bovine serum albumin. The unselected phage library (New England Biolabs) was then added. Phage panning, clone isolation, DNA sequencing, and bioinformatic analyses were performed as described previously (24Carter D.M. Gagnon J.N. Damlaj M. Mandava S. Makowski L. Rodi D.J. Pawelek P.D. Coulton J.W. J. Mol. Biol. 2006; 357: 236-251Crossref PubMed Scopus (32) Google Scholar). Dynamic Light Scattering—Light scattering was measured from purified TonB and FhuD dialyzed twice (18 h, 4 °C) in 100 mm Hepes, pH 7.4, containing 150 mm NaCl. Purified [Fhu switch-MBP] fusion protein (24Carter D.M. Gagnon J.N. Damlaj M. Mandava S. Makowski L. Rodi D.J. Pawelek P.D. Coulton J.W. J. Mol. Biol. 2006; 357: 236-251Crossref PubMed Scopus (32) Google Scholar) (containing FhuA residues 21AWGPAAT27 fused to the N terminus of maltose-binding protein) and BSA were dialyzed against the same buffer and used in DLS measurements as positive and negative controls, respectively. TonB (4.0 μm) and FhuD (3.0 μm) were separately analyzed as discrete scattering species. Similarly, BSA (1.5 μm) and [Fhu switch-MBP] (2.5 μm) were analyzed separately. For a 1:1 molar ratio of TonB to FhuD, each protein at 1.7 μm was mixed prior to recording DLS readings. For 1:1 mixtures of TonB with BSA or with [Fhu switch-MBP], proteins were each at 1.0 μm. Protein mixtures were incubated for 30 min at room temperature prior to centrifugation and analysis. Data acquisition was performed in a 12-μl quartz cuvette at 20 °C using a temperature-controlled DynaPro E-50-830 dynamic light scattering instrument (Protein Solutions, Charlottesville, VA). The scattering signal was measured at a wavelength of 824.9 nm and an angle of 90°. Data were collected for 7000 s with a 10-s averaging time and replicated with two independent protein preparations. From the Dynamics version 6.3.18 software (Protein Solutions, Charlottesville, VA), data were filtered (base line <1.01 and sum of squares <500) before exporting to Sedfit version 9.3. Analyses of hydrodynamic radii (Rh) were performed using the continuous intensity distribution model (25Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (2978) Google Scholar) in Sedfit at a resolution of 100 for radii between 1 and 50 nm. Buffer densities and viscosities were set to 1.00442 and 0.01065, respectively, as determined by Sednterp version 1.08. All values of Rh from the Dynamics software exhibited less than 14% polydispersity, except for the TonB-FhuD mixture and [Fhu switch-MBP] (21 and 19%, respectively). For Sedfit analyses of discrete noninteracting species (DNS), autocorrelation data sets were imported from the Dynamics software package and fit to a single species field autocorrelation function. Values of s for TonB were determined previously (20Khursigara C.M. De Crescenzo G. Pawelek P.D. Coulton J.W. J. Biol. Chem. 2004; 279: 7405-7412Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Using analytical ultracentrifugation, we determined by sedimentation velocity experiments sedimentation coefficients for FhuD and the TonB-FhuD complex, 2.27 s and 3.5 s, respectively. From literature reports, s values for MBP (26Yang Y.R. Schachman H.K. Biophys. Chem. 1996; 59: 289-297Crossref PubMed Scopus (7) Google Scholar) and BSA (27Lebowitz J. Lewis M.S. Schuck P. Protein Sci. 2002; 11: 2067-2079Crossref PubMed Scopus (605) Google Scholar) were obtained. All were constrained in DNS analyses. Molecular mass values for discrete scattering species, either uncomplexed TonB, uncomplexed FhuD, or 1:1 heterocomplexes, were initially set to predicted values and then refined by nonlinear regression until r.m.s.d. errors were minimized. In addition to proteins TonB, FhuD, [Fhu switch-MBP], or complexes formed by these proteins, two scattering species were observed; the Dynamics program predicted these uncharacterized species to have hydrodynamic radii of ∼1 and ∼100 nm respectively. Hydrodynamic parameters for these species were factored into DLS analyses to optimize fits to the autocorrelation function. Fluorescence Spectroscopy—The fluorescent dye AEDANS was conjugated to FhuD and FhuD T181C in a reaction buffer of 100 mm Hepes, pH 7.4, 150 mm NaCl. Following reduction of disulfide bonds with a 10-fold molar excess of tris-(2-carboxyethyl)phosphine, dye was added to a 10-fold molar excess. Conjugation proceeded in the dark with stirring for 4 h at room temperature. Reactions were quenched by addition of β-mercaptoethanol. Excess label was removed by exhaustive dialysis against four 1-liter changes of 100 mm Hepes, pH 7.4, containing 150 mm NaCl in the dark at 4 °C. After dialysis, free dye was present at picomolar concentrations. Conjugates were then centrifuged at 18,000 × g for 30 min at 4 °C. Labeled proteins were stored at 4 °C in the dark. Conjugation of FhuD T181C with the dye MDCC was performed as described above except that MDCC was dissolved in Me2SO prior to its addition to protein. Efficiency of labeling (mol dye:mol protein) was calculated from absorption data using the following tabulated (Invitrogen) molar extinction coefficients: 5700 m-1 cm-1 at 336 nm for AEDANS and 50,000 m-1 cm-1 at 419 nm for MDCC and from protein concentrations as determined by protein assays. Fluorescence data were collected with a Varian Cary Eclipse fluorescence spectrophotometer. Emission spectra were recorded at excitation and emission wavelengths of 280 and 340 nm, respectively, for intrinsic fluorescence measurements; at 336 and 490 nm, respectively, for AEDANS-labeled FhuD and FhuD T181C; and at 419 and 466 nm, respectively, for MDCC-labeled FhuD T181C. Excitation and emission slits were set between 2.5 and 5 nm and 5 and 10 nm, respectively. Measurements were taken in triplicate at 20 °C. Data were corrected for changes in fluorescence intensity attributed to dilution of protein and the minimal fluorescence contributions of Fcn, TonB, and buffer (100 mm Hepes, pH 7.4, 150 mm NaCl). Binding of Fcn to either FhuD (1.5 μm) or FhuD T181C (0.5 μm) was monitored by recording the fluorescence emission after additions of Fcn up to a 10-fold molar excess. For each data point, Fcn was added from a stock solution, and after 3 min of incubation, the change in fluorescence was recorded. Titrations of labeled conjugates with either Fcn or TonB were conducted in an identical manner. Fluorescence quenching was expressed as the percentage decrease in fluorescence upon ligand addition compared with the theoretical maximum whereby quenching would result in complete loss of fluorescence. Data were fit (Sigmaplot) to an equation describing a rectangular hyperbola using the single binding site model or to a sum of two hyperbolics using the model that describes two independent binding sites. Surface Plasmon Resonance (SPR)—Binding interactions between TonB and FhuD or between Cys-TonB and FhuD were examined in real time using BIAcore 2000/3000 instrumentation with research grade CM4 sensor chips (BIAcore AB, Uppsala, Sweden). Experiments were performed in triplicate at 25 °C using filtered (0.2 μm) and degassed HBS-ET (50 mm Hepes, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.05% (v/v) Tween 20). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide, and 2-(2-pyridinyldithio)ethaneamine were from BIAcore AB. Protein grade detergents (10% Tween 20, 10% Triton X-100, 30% Empigen) were from Calbiochem. All other chemicals were reagent grade quality. For amine coupling, TonB was immobilized according to a standard BIAcore protocol. For ligand thiol-coupling, 20 μl of freshly mixed solution I (200 mm 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 50 mm N-hydroxysuccinimide in water) was injected (5 μl/min) over the sensor chip activating carboxymethyl groups to reactive esters. Reactive thiol groups were then introduced by a 30-μl injection of freshly prepared solution II (80 mm 2-(2-pyridinyldithio) ethaneamine in 0.1 m sodium borate, pH 8.5). Diluted Cys-TonB ligand (3 μg/ml in 10 mm sodium acetate, pH 4.5) was injected manually until ∼120 RU were bound. Finally, three injections (20 μl) of freshly prepared solution III (50 mm l-cysteine in 0.1 m sodium formate, pH 4.3, containing 1 m NaCl) deactivated excess reactive groups and removed any nonspecifically bound ligand. Coupling efficiencies were typically ∼50%. Reference surfaces were prepared in a similar manner without any ligand addition. Immobilized TonB and Cys-TonB surfaces were washed overnight at 5 μl/min in running buffer. Prior to use, FhuD analyte was dialyzed against HBS-ET, and immobilized TonB or Cys-TonB surfaces were conditioned at 50 μl/min using regeneration scheme A as follows: two 25-μl injections each of (i) 0.05% (v/v) Empigen, 0.5 m NaCl, 50 mm EDTA, 10 mm NaOH in HBS-ET, (ii) 0.05% (v/v) Triton X-100, 0.5 m NaCl, 50 mm EDTA, 10 mm NaOH in HBS-ET, and (iii) HBS-ET. For kinetic experiments, FhuD (0.1-1 μm in the absence and presence of a 10-fold molar excess of Fcn) was injected at 50 μl/min (120 s association + 120 s dissociation) over amine-coupled TonB or thiol-coupled Cys-TonB. Surface performance and mass transfer tests confirmed that the ligand density and regeneration conditions were appropriate. All acquired data were double-referenced (28Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (644) Google Scholar) and analyzed globally according to the simple 1:1 binding model (A + B = AB) or to the heterogeneous ligand model in the BIAevaluation 4.1 software (BIAcore AB). Kinetic estimates represent fits to the experimental data with χ2 values below 1. Multicomponent SPR analyses between FhuA, TonB, and FhuD were performed. Initially, amine-coupled TonB surfaces (250 RU) or thiol-coupled Cys-TonB surfaces (100 RU) were prepared. Then, either a TonB-FhuA or a TonB-FhuD binary complex was formed by injecting each analyte at 50 μl/min. By injecting FhuD over TonB-FhuA complexes or by injecting FhuA over TonB-FhuD complexes, ternary complex formation was assessed. Identification of TonB-binding Sites on FhuD by Phage Display—Following affinity selection versus immobilized TonB (24Carter D.M. Gagnon J.N. Damlaj M. Mandava S. Makowski L. Rodi D.J. Pawelek P.D. Coulton J.W. J. Mol. Biol. 2006; 357: 236-251Crossref PubMed Scopus (32) Google Scholar), 135 unique disulfide-constrained peptides from the Ph.D.-C7C library and 105 unique linear peptides from the Ph.D.-12 library were analyzed. These phage-displayed peptides were scanned for their similarity to the primary sequence of FhuD using the receptor ligand contacts (RELIC) program RELIC/MATCH (29Mandava S. Makowski L. Devarapalli S. Uzubell J. Rodi D.J. Proteomics. 2004; 4: 1439-1460Crossref PubMed Scopus (103) Google Scholar). Among these sequences, 8 from the Ph.D.-C7C library (Table 1) and 13 from the Ph.D.-12 library (Table 2) were found to share similarities with the primary sequence of FhuD. The Ph.D.-C7C and Ph.D.-12 sequences were observed (Fig. 1) to cluster at four discrete r" @default.
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