Title: Enhanced Binding of TonB to a Ligand-loaded Outer Membrane Receptor
Abstract: The ferric hydroxymate uptake (FhuA) receptor from Escherichia coli facilitates transport of siderophores ferricrocin and ferrichrome and siderophore-antibiotic conjugates such as albomycin and rifamycin CGP 4832. FhuA is also the receptor for phages T5, T1, Φ80, UC-1, for colicin M and for the antimicrobial peptide microcin MccJ21. Energy for transport is provided by the cytoplasmic membrane complex TonB·ExbB·ExbD, which uses the proton motive force of the cytoplasmic membrane to transduce energy to the outer membrane. To accomplish energy transfer, TonB contacts outer membrane receptors. However, the stoichiometry of TonB· receptor complexes and their sites of interaction remain uncertain. In this study, analyses of FhuA interactions with two recombinant TonB proteins by analytical ultracentrifugation revealed that TonB forms a 2:1 complex with FhuA. The presence of the FhuA-specific ligand ferricrocin enhanced the amounts of complex but is not essential for its formation. Surface plasmon resonance experiments demonstrated that FhuA·TonB interactions are multiple and have apparent affinities in the nanomolar range. TonB also possesses two distinct binding regions: one in the C terminus of the protein, for which binding to FhuA is ferricrocin-independent, and a higher affinity region outside the C terminus, for which ferricrocin enhances interactions with FhuA. Together these experiments establish that FhuA·TonB interactions are more intricate than originally predicted, that the TonB·FhuA stoichiometry is 2:1, and that ferricrocin modulates binding of FhuA to TonB at regions outside the C-terminal domain of TonB. The ferric hydroxymate uptake (FhuA) receptor from Escherichia coli facilitates transport of siderophores ferricrocin and ferrichrome and siderophore-antibiotic conjugates such as albomycin and rifamycin CGP 4832. FhuA is also the receptor for phages T5, T1, Φ80, UC-1, for colicin M and for the antimicrobial peptide microcin MccJ21. Energy for transport is provided by the cytoplasmic membrane complex TonB·ExbB·ExbD, which uses the proton motive force of the cytoplasmic membrane to transduce energy to the outer membrane. To accomplish energy transfer, TonB contacts outer membrane receptors. However, the stoichiometry of TonB· receptor complexes and their sites of interaction remain uncertain. In this study, analyses of FhuA interactions with two recombinant TonB proteins by analytical ultracentrifugation revealed that TonB forms a 2:1 complex with FhuA. The presence of the FhuA-specific ligand ferricrocin enhanced the amounts of complex but is not essential for its formation. Surface plasmon resonance experiments demonstrated that FhuA·TonB interactions are multiple and have apparent affinities in the nanomolar range. TonB also possesses two distinct binding regions: one in the C terminus of the protein, for which binding to FhuA is ferricrocin-independent, and a higher affinity region outside the C terminus, for which ferricrocin enhances interactions with FhuA. Together these experiments establish that FhuA·TonB interactions are more intricate than originally predicted, that the TonB·FhuA stoichiometry is 2:1, and that ferricrocin modulates binding of FhuA to TonB at regions outside the C-terminal domain of TonB. Gram-negative bacteria have evolved efficient acquisition systems for the uptake of scarce nutrients. One of the most sought after nutrients is iron, an element whose bioavailability is limited at physiological conditions, because it forms insoluble ferric hydroxides. Escherichia coli satisfies its iron requirements by expressing high affinity receptors at its cell surface. These proteins bind and transport iron-chelating siderophores from the external milieu into the periplasm in an energy-dependent manner. One such high affinity uptake system relies upon FhuA, receptor for the hydroxamate siderophore ferricrocin (Fc). 1The abbreviations used are: Fc, ferricrocin; AUC, analytical ultracentrifugation; SPR, surface plasmon resonance; RU, resonance units; CM, cytoplasmic membrane; OM, outer membrane; LDAO, N-lauryldimethylamine oxide; CT, C-terminal; Mb, buoyant molecular mass; r.m.s.d., root mean square deviation; vbar, partial specific volume. FhuA also facilitates the transport of siderophoreantibiotic conjugates such as albomycin and rifamycin CGP 4832, is the receptor for phages T5, T1, Φ80, UC-1, for colicin M and for the antimicrobial peptide microcin MccJ21 (1Braun V. Hantke K. Köster W. Sigel A. Sigel H. Metal Ions in Biological Systems. 35. Marcel Dekker, Inc., New York1998: 67-145Google Scholar, 2Moeck G.S. Coulton J.W. Mol. Microbiol. 1998; 28: 675-681Crossref PubMed Scopus (248) Google Scholar, 3Ferguson A.D. Coulton J.W. Diederichs K. Welte W. Messerschmidt A. Huber R. Poulos T. Wieghardt K. Handbook of Metalloproteins. John Wiley & Sons, Ltd., Chichester2001: 834-849Google Scholar). Energy for siderophore transport is provided by the cytoplasmic membrane (CM) complex TonB·ExbB·ExbD, which exploits the electrochemical potential from the proton motive force of the CM and transduces energy to the outer membrane (OM) (4Braun V. Braun M. Curr. Opin. Microbiol. 2002; 5: 194-201Crossref PubMed Scopus (162) Google Scholar). Although many proteins involved in TonB-dependent siderophore transport have been identified and studied, the molecular mechanism of siderophore uptake from the external environment to the periplasm of the bacteria remains obscure. Evidence for conformational changes occurring in FhuA following siderophore binding was provided by differential recognition by monoclonal antibodies of ligand-free versus ligand-loaded receptor (5Moeck G.S. Tawa P. Xiang H. Ismail A.A. Turnbull J.L. Coulton J.W. Mol. Microbiol. 1996; 22: 459-471Crossref PubMed Scopus (62) Google Scholar). At the atomic level, x-ray crystallographic structures of FhuA (6Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (673) Google Scholar, 7Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar), and the receptor FecA (8Ferguson A.D. Chakraborty R. Smith B.S. Esser L. van der Helm D. Deisenhofer J. Science. 2002; 295: 1715-1719Crossref PubMed Scopus (307) Google Scholar) displayed conspicuous structural changes upon ligand binding. A switch helix (residues 24–29) on the periplasmic face of FhuA unwound to a random coil, and there was a 17-Å translocation of the extreme N terminus, proximal to the Ton box of FhuA. This conformational change was proposed to be a signal reporting the ligand-loaded status of FhuA to TonB (6Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (673) Google Scholar, 7Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). However, two other OM receptors, FepA and BtuB (9Buchanan S.K. Smith B.S. Venkatramani L. Xia D. Esser L. Palnitkar M. Chakraborty R. van der Helm D. Deisenhofer J. Nat. Struct. Biol. 1999; 6: 56-63Crossref PubMed Scopus (495) Google Scholar, 10Chimento D.P. Kadner R.J. Wiener M.C. J. Mol. Biol. 2003; 332: 999-1014Crossref PubMed Scopus (67) Google Scholar), whose structures have been published at atomic resolution, do not contain a switch helix. The unwinding of a switch helix is therefore not a common mechanistic feature for all OM receptors. In vivo cross-linking studies of TonB and FepA provided the first biochemical evidence of interactions between TonB and OM receptors (11Skare J.T. Ahmer B.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). These results corroborated genetic analyses where point mutations in the Ton box of FhuA were suppressed by mutations in the tonB gene, suggesting a functional interaction near Gln160 of TonB (12Schoffler H. Braun V. Mol. Gen. Genet. 1989; 217: 378-383Crossref PubMed Scopus (150) Google Scholar, 13Gunter K. Braun V. FEBS Lett. 1990; 274: 85-88Crossref PubMed Scopus (71) Google Scholar). Additional cross-linking studies demonstrated that specific ligands of the OM receptors enhance their association with TonB (14Moeck G.S. Coulton J.W. Postle K. J. Biol. Chem. 1997; 272: 28391-28397Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Moeck G.S. Letellier L. J. Bacteriol. 2001; 183: 2755-2764Crossref PubMed Scopus (56) Google Scholar, 16Cadieux N. Kadner R.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10673-10678Crossref PubMed Scopus (146) Google Scholar). Detailed analyses of TonB·BtuB interactions by site-directed spin labeling revealed that TonB requires a specific orientation for functional contact with the Ton box. Changes in conformation in the Ton box region caused by proline substitutions abrogated transport of the ligand (17Cadieux N. Bradbeer C. Kadner R.J. J. Bacteriol. 2000; 182: 5954-5961Crossref PubMed Scopus (73) Google Scholar). The crystal structure of C-terminal TonB (residues 164–239) provided first evidence that TonB forms a dimer (18Chang C. Mooser A. Pluckthun A. Wlodawer A. J. Biol. Chem. 2001; 276: 27535-27540Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To date, two models for TonB-OM receptor interaction have been proposed. In what has been termed the propeller model (19Postle K. Kadner R.J. Mol. Microbiol. 2003; 49: 869-882Crossref PubMed Scopus (253) Google Scholar), two TonB monomers are intertwined, interacting with OM receptors. This model suggested that dimerized TonB undergoes rotary motion, similar to the mechanism described for the bacterial flagellar motor that is powered by MotA and MotB. ExbB and ExbD are homologous to MotA and MotB (20Zhai Y.F. Heijne W. Saier Jr., M.H. Biochim. Biophys. Acta. 2003; 1614: 201-210Crossref PubMed Scopus (43) Google Scholar). An alternate model describes the shuttling of TonB between the CM and OM (19Postle K. Kadner R.J. Mol. Microbiol. 2003; 49: 869-882Crossref PubMed Scopus (253) Google Scholar, 21Larsen R.A. Letain T.E. Postle K. Mol. Microbiol. 2003; 49: 211-218Crossref PubMed Scopus (48) Google Scholar). The shuttle model is supported by in vivo labeling experiments that demonstrate periplasmic accessibility of the extreme N terminus of TonB to the cysteine-specific marker Oregon Green 488 maleimide. According to this model, TonB in complex with ExbB and ExbD in the CM are in an unenergized conformation. ExbB·ExbD use the proton motive force to energize TonB, allowing its C-terminal portion to interact with the OM. This may cause the release of the N terminus of TonB from the CM and transfer of stored potential energy from TonB to OM receptors, thereby facilitating ligand import. To analyze interactions between FhuA and TonB, we selected two complementary biophysical methods. Analytical ultracentrifugation (AUC) was used to determine the buoyant molecular weights and stoichiometries of two genetically engineered, soluble derivatives of TonB: a hexahistidine-tagged full-length TonB (H6.′TonB) consisting of residues 32–239 of the mature protein; and a hexahistidine-tagged C-terminal TonB (H6.′TonB (CT)), residues 155–239, both purified to homogeneity. Sedimentation velocity experiments were conducted for the two TonBs and for FhuA, either individually or as protein·protein complexes. Surface plasmon resonance (SPR) optical biosensors (Biacore) were used to derive thermodynamic parameters of the interacting proteins. Our results demonstrate that the TonB·FhuA stoichiometry is 2:1, that TonB interacts with FhuA in an Fc-independent manner, that, in addition to its C-terminal portion, the N-terminal region of TonB participates in binding to FhuA, and that Fc modulates interactions between FhuA and the N-terminal region of TonB. Strains—Escherichia coli AW740 harbors plasmid pHX405 and expresses recombinant FhuA with a hexahistidine tag at position 405 (22Ferguson A.D. Breed J. Diederichs K. Welte W. Coulton J.W. Protein Sci. 1998; 7: 1636-1638Crossref PubMed Scopus (43) Google Scholar). E. coli ER2566, transformed with pET28 plasmid containing H6.′TonB, was similar to the construct described by Moeck and Letellier (15Moeck G.S. Letellier L. J. Bacteriol. 2001; 183: 2755-2764Crossref PubMed Scopus (56) Google Scholar) and was corrected to reflect the wild-type sequences of residues 32–239 of TonB. In addition, E. coli ER2566 was the host strain for pET28 into which was cloned the gene for the C terminus of TonB (residues 155–239). All plasmids were confirmed for their sequence fidelity by DNA sequencing at Sheldon Biotechnology Center, McGill University. Protein Purification—FhuA was purified (6Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (673) Google Scholar, 22Ferguson A.D. Breed J. Diederichs K. Welte W. Coulton J.W. Protein Sci. 1998; 7: 1636-1638Crossref PubMed Scopus (43) Google Scholar) in N-lauryldimethylamine oxide (LDAO; Fluka) and detergent-exchanged using Q-Sepharose anion exchange media (Amersham Biosciences) into 100 mm HEPES (pH 7.4), 150 mm NaCl, 0.1% Tween 20 (Calbiochem), hence-forth designated Biacore running buffer. H6.′TonB and H6.′TonB (CT) were purified using Ni2+-nitrilotriacetic acid Superflow resin (Qiagen) followed by cation exchange on SP-Sepharose (Amersham Biosciences). Prior to SPR experiments, each TonB protein was dialyzed into Biacore running buffer. To remove the hexahistidine tag, H6.′TonB was incubated with thrombin protease (Amersham Biosciences): 1 unit of protease per mg of H6.′TonB. After 3 h at ambient temperature, the reaction mixture was applied to a Ni2+-nitrilotriacetic acid column, capturing uncleaved H6.′TonB; the flow-through was applied to an SP-Sepharose column. Eluted protein was assayed by Western blotting with an anti-His6 monoclonal antibody (Cedarlane Laboratories Limited, Mississauga, Ontario, Canada) for the absence of the hexahistidine tag. TonB-reactive protein was confirmed by a cross-reactive monoclonal antibody that was raised against Trypanosoma brucei procyclin (CLP001A, Cedarlane) and that recognizes the proline-glutamic acid repeat portion of TonB protein. Protein concentrations were determined using the protein dye binding assay (Bio-Rad) and BCA assay (Pierce). Analytical Ultracentrifugation—Samples were prepared for AUC by extensive dialysis against an AUC buffer: 100 mm HEPES (pH 8.0), 150 mm NaCl. In some experiments, AUC buffer was supplemented with either LDAO at 0.3% or n-octyltetraoxyethylene (C8E4; Bachem) at 0.4%. Protein concentrations were adjusted between 0.3 and 0.9 mg/ml. Ligand-loaded receptor was prepared by mixing Fc and FhuA in a 10:1 molar ratio, 30 min at room temperature. Excess Fc was removed by dialysis against AUC buffer using a Spectrapor dialysis membrane (POR-6, 25-kDa cutoff). Sedimentation velocity experiments were performed on all protein samples using a Beckman XL-I Analytical Ultracentrifuge. The sample and the reference sectors of 1.2-cm path length double-sector ultracentrifuge cells were filled with 400 μl of protein and AUC buffer, respectively. All sedimentation velocity runs were performed at 40,000 rpm, with absorbance scans monitored at 280 nm in 10-min intervals over a total spin time of 4 h at 24.6 °C. Protein complexes were formed immediately prior to each spin by mixing H6.′TonB:FhuA at 1:1 and at 2:1 molar ratios, or by mixing H6.′TonB (CT):FhuA at 4:1 and at 8:1 molar ratios. In both cases, H6.′TonB and H6.′TonB (CT) were varied relative to a fixed concentration of FhuA or FhuA plus Fc. Analysis of AUC Data—Sedimentation velocity data were analyzed by the computer program SEDFIT (23Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3150) Google Scholar). Initial sedimentation profiles were obtained by fitting the data to the Continuous c(S) Model. Global frictional ratios determined by c(S) analysis were allowed to float to convergence. Buoyant molecular weight (Mb) and final s values were determined using the discrete non-interacting species model of SEDFIT. In sedimentation experiments involving LDAO, the detergent was modeled as a known sedimenting species with an s value of –0.1 and a micellar buoyant molecular mass of 2700 Da. Sedimentation coefficients of uncomplexed FhuA, H6.′TonB, and H6.′TonB (CT) were initially estimated from c(S) analyses and then refined by direct fitting to the Lamm equation using the non-interacting discrete species model of SEDFIT. Initial Mb values were obtained from the known molecular weights of each protein. Both s and Mb were allowed to float to convergence using the non-linear regression algorithms of SEDFIT. Sedimentation velocity data of mixtures of FhuA plus H6.′TonB and FhuA plus H6.′TonB (CT) were fit to the discrete non-interacting species model using constrained analysis. From spins of the individual species, initial sedimentation parameters (s and Mb) of uncomplexed components (s1 and s2) were entered. The initial sedimentation parameters of predicted complexes (s3) were input based on the predicted Mb as integral additions of the Mb values of s1 and s2; initial sedimentation coefficients of s3 were estimated from c(S) analyses. The sedimentation parameters of s3 were then floated to convergence, whereas s1 and s2 parameters remained constrained. Fits to the model were evaluated on the basis of the distribution of residuals and r.m.s.d. errors. Optimization of the fits was performed by varying stoichiometric combinations of s1, s2, and s3 and repeating the constrained analyses until random distributions of residuals and minimal r.m.s.d. errors were obtained. As an unbiased test, data from mixtures of FhuA plus H6.′TonB and FhuA plus H6.′TonB (CT) were fit to the non-interacting discrete species model, assuming the presence of species with sedimentation parameters equivalent to uncomplexed s1 and s2. The r.m.s.d. errors and residuals were compared with those obtained from optimized fits in which the presence of s3 was modeled. Immobilization of Recombinant TonB Proteins on Biacore Sensor Chips—Several sensor chip surfaces were assessed for binding of TonB proteins and for their regeneration, including CM4 (formerly B1), CM5, nitrilotriacetic acid (NTA), C1, and F1. Optimally, solutions of H6.′TonB (150 nm) and H6.′TonB (CT) (1.8 μm) in 10 mm acetate buffer (pH 5.5) were used to covalently immobilize the proteins on CM4 sensor chips using amine coupling procedures via N-hydroxysuccinimide/N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride; deactivation was accomplished with ethanolamine (24Johnsson B. Lofas S. Lindquist G. Anal. Biochem. 1991; 198: 268-277Crossref PubMed Scopus (1213) Google Scholar). To prepare control surfaces, activation and deactivation procedures were performed in the absence of TonB proteins. Preliminary and Steady-state Assays by SPR—SPR measurements were performed using a Biacore 2000 and a Biacore 3000 (Biacore AB) and were carried out in triplicate at 25 °C. The data collection rate was set to 10 Hz. Biacore running buffer was used to dilute FhuA. Steady-state experiments were conducted with a flow rate of 5 μl/min. To reach steady-state equilibrium, the injection time was 1200 s followed by injections of buffer for 240 s. Regeneration was achieved by three pulses (1 min) of 5 mm NaOH, 0.1% Tween 20, and a subsequent EXTRA-CLEAN procedure. Biacore Data Preparation and Analysis—Data were prepared using the double referencing method (25Rich R.L. Myszka D.G. Curr. Opin. Biotechnol. 2000; 11: 54-61Crossref PubMed Scopus (590) Google Scholar). For global analysis, the sensorgrams were transformed to concentration units using the molecular weights of injected proteins. All curves were reduced to 700 evenly spaced sampling points. For each set of individual curves corresponding to injections of various concentrations of FhuA over the same surface, global fitting was carried out using a simple Langmuirian model in the SPRevolution software package of De Crescenzo and colleagues (26De Crescenzo G. Grothe S. Lortie R. Debanne M.T. O'Connor-McCourt M. Biochemistry. 2000; 39: 9466-9476Crossref PubMed Scopus (48) Google Scholar, 27De Crescenzo G. Grothe S. Zwaagstra J. Tsang M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 29632-29643Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). For steady-state analysis, the apparent thermodynamic dissociation constants were determined by plotting the control corrected plateau value (Req) versus the injected concentration of FhuA. In the case of H6.′TonB, the Kd values were derived by fitting the experimental Req values to a model of two independent population interactions, Req=Rmax1×(C/(C+Kd1))+Rmax2×(C/(C+Kd2)) where C corresponds to the injected FhuA concentration, Rmax1 and Rmax2 correspond to the maximal amount of FhuA that can be bound to each active H6.′TonB population, and Kd1 and Kd2 to their respective thermodynamic dissociation constants. Alternatively for H6.′TonB (CT), the experimental data were adequately fit with a simple interaction model in Equation 2 as follows. Req=Rmax×(C/(C+Kd))(Eq. 1) The fitting procedure was performed in Microsoft Excel by non-linear regression with Rmax values and Kd values as floating parameters. Analytical Ultracentrifugation—Sedimentation velocity experiments on FhuA, H6.′TonB, and H6.′TonB (CT), individually and in complex, were performed in buffers containing the neutrally buoyant detergent C8E4 such that actual molecular weight values of the protein components could be determined. In advance of determining the stoichiometry of FhuA·TonB complexes, sedimentation parameters of the individual uncomplexed species were determined and used as prior knowledge in the analysis of sedimenting complexes. To obtain the best Mr estimates of the sedimenting species, apparent vbar (Φ) values were determined from the known molecular weights of each protein, along with the buoyant molecular weights (Mb) determined by fitting to the non-interacting discrete species model of SEDFIT. As observed by Boulanger et al. (28Boulanger P. le Maire M. Bonhivers M. Dubois S. Desmadril M. Letellier L. Biochemistry. 1996; 35: 14216-14224Crossref PubMed Scopus (90) Google Scholar), their experimentally determined vbar of FhuA (0.776 ml/g) is significantly different from the value predicted from the amino acid sequence of the protein (29Perkins S.J. Eur. J. Biochem. 1986; 157: 169-180Crossref PubMed Scopus (549) Google Scholar). Similarly, using the known Mr of FhuA and the experimental Mb, we determine a Φ of 0.775 ml/g (Table I), in contrast to the sequence-predicted value of 0.735. The Φ values were also determined for H6.′TonB and H6.′TonB (CT), and these values (0.719 and 0.714 ml/g; Table I) also differ from vbars predicted from amino acid sequences (0.740 and 0.730 ml/g, respectively). The fits of the sedimentation velocity data of uncomplexed species to the non-interacting discrete species model were in all cases excellent with random distributions of residuals and least-squares (r.m.s.d.) errors of fit equal to or less than 0.0061.Table ISedimentation velocity parameters from uncomplexed FhuA, H6.′TonB, and H6.′TonB (CT)ProteinΦaΦ = (1 - Mb/Mseq)/r.MbbBuoyant molecular weight determined from fit to a non-interacting discrete species model.MrcMr = Mb/(1 - Φ).MseqdMseq = molecular weight calculated from primary amino acid sequence.sr.m.s.d.er.m.s.d. = least squares error of the fit to the non-interacting discrete species model.f/foff/fo = frictional ratio.ml/gDaFhuA0.77518,44081,95580,0003.500.00521.76FhuA plus Fc0.77518,43081,91180,0003.600.00611.76H6.′TonB0.7196,98927,43024,9001.400.00362.39H6.′TonB (CT)0.7146,80023,09011,9031.620.00351.96a Φ = (1 - Mb/Mseq)/r.b Buoyant molecular weight determined from fit to a non-interacting discrete species model.c Mr = Mb/(1 - Φ).d Mseq = molecular weight calculated from primary amino acid sequence.e r.m.s.d. = least squares error of the fit to the non-interacting discrete species model.f f/fo = frictional ratio. Open table in a new tab Given these Φ values, FhuA sedimented as a monomeric protein with a molecular mass of ∼80,000 Da; the presence of bound Fc resulted in a small yet reproducible increase in the sedimentation coefficient from 3.50 to 3.60 s, possibly due to structural rearrangements in the protein upon binding to ligand as has been observed in many of the crystal structures of bacterial OM receptors (6Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (673) Google Scholar, 7Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 8Ferguson A.D. Chakraborty R. Smith B.S. Esser L. van der Helm D. Deisenhofer J. Science. 2002; 295: 1715-1719Crossref PubMed Scopus (307) Google Scholar, 9Buchanan S.K. Smith B.S. Venkatramani L. Xia D. Esser L. Palnitkar M. Chakraborty R. van der Helm D. Deisenhofer J. Nat. Struct. Biol. 1999; 6: 56-63Crossref PubMed Scopus (495) Google Scholar, 30Chimento D.P. Mohanty A.K. Kadner R.J. Wiener M.C. Nat. Struct. Biol. 2003; 10: 394-401Crossref PubMed Scopus (239) Google Scholar). H6.′TonB sedimented as a monomer in C8E4-containing AUC buffer with a Mr of 27,430. H6.′TonB (CT) sedimented as a dimer with a Mr of 23,090 (Table I). Using the same data sets, analyses modeling the presence of additional oligomeric states of either TonB species resulted in extremely poor fits to the data (data not shown). Of the three proteins analyzed, c(S) analysis indicates that H6.′TonB has the highest frictional ratio: f/fo = 2.39. This is consistent with H6.′TonB having an extended conformation in solution. H6.′TonB (CT) has a lower frictional ratio (f/fo = 1.96), because it is a truncated form of H6.′TonB; furthermore, H6.′TonB (CT) is dimeric, resulting in the protein having a globular shape as compared with the more extended H6.′TonB. FhuA and the two TonB species were then mixed together for sedimentation velocity ultracentrifugation and were analyzed for the formation of complexes: FhuA plus H6.′TonB (CT) and FhuA plus H6.′TonB, both in the absence and presence of Fc. Sedimentation data were analyzed by first fitting to a continuous c(S) model to obtain initial sedimentation parameters. This was followed (Table II) by direct fitting to the Lamm equation by a non-interacting discrete species model using the c(S)-derived sedimentation parameters and known molecular weights of individual proteins as prior knowledge (Table I). Refinement of c(S)-derived sedimentation parameters by the non-interacting species model resulted in converged s values similar to those obtained from the c(S) analysis, indicating a good correlation between the two models. However, unlike c(S) analysis, which employs a global frictional coefficient, the non-interacting species model resolves proteins of different diffusion coefficients, allowing for the calculation of buoyant molecular weight (Mb) for each sedimenting species. In the context of AUC, the term “non-interacting species” refers to sedimenting species that do not reversibly interact over the time frame of the experiment and can include stable protein complexes. In our case, a complex of FhuA and H6.′TonB (CT) or of FhuA and H6.′TonB is considered “non-interacting” if it is observed to sediment as a single species with a molecular weight that is additive of the uncomplexed components and with a sedimentation coefficient that does not significantly change as the ratio of the uncomplexed components is varied.Table IISedimentation velocity parameters of FhuA plus H6.′TonB and FhuA plus H6.′TonB (CT) complexes determined by fits to a non-interacting discrete species modelFhuAH6.′TonBH6.′TonB (CT)Fcs1Mb1s2Mb2s3Mb3ΦavgaΦavg = weight-averaged apparent vbar of complex.Mr3s1/s2/s3r.m.s.dDaDaDaml/gDa% ctot14-(3.50)bConstrained parameters are indicated by parentheses.(18,440)(1.62)(6,800)4.4724,1770.761101,15855/18/270.005718-(3.50)(18,440)(1.62)(6,800)4.5325,3360.761106,00829/31/330.004914+(3.60)(18,430)(1.62)(6,800)4.6924,0990.761100,83238/08/400.005118+(3.60)(18,430)(1.62)(6,800)4.4425,2500.761105,64837/21/420.006411-(3.50)(18,440)(1.40)(6,989)3.2433,4030.762140,34871/05/150.008012-(3.50)(18,440)(1.40)(6,989)3.2731,7360.762133,34466/17/170.005711+--1.90(13,978)3.5331,2010.762131,09600/10/520.004512+--1.90(13,978)3.4231,2980.762131,50400/20/500.0058a Φavg = weight-averaged apparent vbar of complex.b Constrained parameters are indicated by parentheses. Open table in a new tab Experiments were performed with FhuA (–/+ Fc) mixed with either TonB species such that immediately prior to each centrifugation, proteins were combined at selected molar ratios of FhuA:H6.′TonB (CT) (monomer) = 1:4 and 1:8, or FhuA: H6.′TonB (monomer) = 1:1 and 1:2. Sedimentation velocity ultracentrifugation of these mixtures resulted in a combination of complexed species and uncomplexed species, which could be resolved by analysis of the sedimentation boundaries over the time course of the experiment. Sedimentation velocity data of both FhuA plus H6.′TonB (CT) and FhuA plus H6.′TonB mixtures fit well to the discrete non-interacting species model (Table II and Fig. 1, A–D). In addition to observing sedimenting species corresponding to uncomplexed FhuA (s1) and H6.′TonB (CT) or H6.′TonB (s2), a third sedimenting species (s3) corresponding to FhuA·TonB complex was always observed in the absence and presence of Fc. Modeling interactions without s3 led to dramatically poore