Title: Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli
Abstract: FhuA belongs to a family of specific siderophore transport systems located in the outer membrane of Escherichia coli. The energy required for the transport process is provided by the proton motive force of the cytoplasmic membrane and is transmitted to FhuA by the protein TonB. Although the structure of full-length TonB is not known, the structure of the last 77 residues of a fragment composed of the 86 C-terminal amino acids was recently solved and shows an intertwined dimer (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) J. Biol. Chem. 276, 27535–27540). We analyzed the ability of truncated C-terminal TonB fragments of different lengths (77, 86, 96, 106, 116, and 126 amino acid residues, respectively) to bind to the receptor FhuA. Only the shortest TonB fragment, TonB-77, could not effectively interact with FhuA. We have also observed that the fragments TonB-77 and TonB-86 form homodimers in solution, whereas the longer fragments remain monomeric. TonB fragments that bind to FhuA in vitro also inhibit ferrichrome uptake via FhuA in vivo and protect cells against attack by bacteriophage Φ80. FhuA belongs to a family of specific siderophore transport systems located in the outer membrane of Escherichia coli. The energy required for the transport process is provided by the proton motive force of the cytoplasmic membrane and is transmitted to FhuA by the protein TonB. Although the structure of full-length TonB is not known, the structure of the last 77 residues of a fragment composed of the 86 C-terminal amino acids was recently solved and shows an intertwined dimer (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) J. Biol. Chem. 276, 27535–27540). We analyzed the ability of truncated C-terminal TonB fragments of different lengths (77, 86, 96, 106, 116, and 126 amino acid residues, respectively) to bind to the receptor FhuA. Only the shortest TonB fragment, TonB-77, could not effectively interact with FhuA. We have also observed that the fragments TonB-77 and TonB-86 form homodimers in solution, whereas the longer fragments remain monomeric. TonB fragments that bind to FhuA in vitro also inhibit ferrichrome uptake via FhuA in vivo and protect cells against attack by bacteriophage Φ80. The cell wall of Gram-negative bacteria consists of two lipid bilayers, the outer membrane and the cytoplasmic membrane enclosing the peptidoglycan layer. A number of different transport pathways regulate the uptake of essential compounds into the cell. One class of outer membrane transporters is connected to the cytoplasmic membrane by the TonB protein; therefore, they are called TonB-dependent receptors. The three-dimensional structure of a short C-terminal fragment of TonB is available in the literature (1Chang C. Mooser A. Pluckthun A. Wlodawer A. J. Biol. Chem. 2001; 276: 27535-27540Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). One of these receptors in Escherichia coli is the ferric hydroxamate uptake system containing the integral outer membrane protein FhuA (2Coulton J.W. Mason P. Cameron D.R. Carmel G. Jean R. Rode H.N. J. Bacteriol. 1986; 165: 181-192Crossref PubMed Google Scholar), which serves as a receptor for the iron siderophore ferrichrome (FC), 1The abbreviations used are: FC, ferrichrome; Cit, ferric dicitrate; NB, nutrient broth; LB, Luria-Bertani media; r.m.s.d., root-meansquare deviation. the antibiotics albomycin and rifamycin CGP 4832, colicin M, and microcin J25, and the phages T1, T5, and Φ80. Other TonB-dependent iron transporters of the outer membrane include FecA for ferric dicitrate (Cit) uptake (3Pressler U. Staudenmaier H. Zimmermann L. Braun V. J. Bacteriol. 1988; 170: 2716-2724Crossref PubMed Google Scholar), FepA for enterobactin uptake (4Lundrigan M.D. Kadner R.J. J. Biol. Chem. 1986; 261: 10797-10801Abstract Full Text PDF PubMed Google Scholar), and BtuB for vitamin B12 uptake (5Heller K. Kadner R.J. J. Bacteriol. 1985; 161: 904-908Crossref PubMed Google Scholar). The transport of all of these ligands requires energy, which is provided by the electrochemical potential of the proton gradient across the cytoplasmic membrane (proton motive force) and is mediated by the protein complexes ExbB, ExbD, and TonB (6Bradbeer C. J. Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (148) Google Scholar, 7Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (155) Google Scholar, 8Postle K. Kadner R.J. Mol. Microbiol. 2003; 49: 869-882Crossref PubMed Scopus (251) Google Scholar). ExbB/D is located in the cytoplasmic membrane, whereas TonB is attached to the membrane by an N-terminal hydrophobic anchor (9Postle K. J. Bioenerg. Biomembr. 1993; 25: 591-601PubMed Google Scholar). The major part of TonB spans the periplasmic space to reach the outer membrane receptors. The crystal structure of FhuA reveals a two domain architecture (10Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar, 11Locher 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 (457) Google Scholar): a β-barrel consisting of 22-antiparallel strands and a globular domain at the N terminus (residues 1–160), called the cork or plug domain filling most of the interior of the barrel. Stability studies using differential scanning calorimetry experiments have shown the autonomous behavior of the cork and the β-barrel that unfold at different temperatures (12Bonhivers M. Desmadril M. Moeck G.S. Boulanger P. Colomer-Pallas A. Letellier L. Biochemistry. 2001; 40: 2606-2613Crossref PubMed Scopus (49) Google Scholar). The interactions between the cork domain and the β-barrel consist of nine salt bridges and more than 60 hydrogen bonds (11Locher 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 (457) Google Scholar). Located at the periplasmic side of FhuA there is a short α-helix, the so-called "switch helix" (residues 24–29). This α-helix has been found to unwind during or following ligand binding, indicating that this structural change might be a signal for TonB to bind FhuA (10Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar, 11Locher 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 (457) Google Scholar). This unwinding was observed in the crystal structures of FhuA with bound ferrichrome (10Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar) or albomycin (13Ferguson A.D. Braun V. Fiedler H.P. Coulton J.W. Diederichs K. Welte W. Protein Sci. 2000; 9: 956-963Crossref PubMed Scopus (118) Google Scholar). On the other hand, the crystal structure of FhuA with the rifamycin derivative CGP-4832 demonstrates that ligand binding causes destabilization rather than unwinding of the switch helix (14Ferguson A.D. Ködding J. Walker G. Bos C. Coulton J.W. Diederichs K. Braun V. Welte W. Structure. 2001; 9: 707-716Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). These structures present a specific ligand binding site that is exposed to the external medium and determined by specific hydrogen bonds between the substrate and residues of both the cork and the β-barrel domain. The crystal structures of FepA (15Buchanan 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 (492) Google Scholar), FecA (16Ferguson A.D. Chakraborty R. Smith B.S. Esser L. van der Helm D. Deisenhofer J. Science. 2002; 295: 1715-1719Crossref PubMed Scopus (304) Google Scholar), and BtuB (17Chimento D.P. Mohanty A.K. Kadner R.J. Wiener M.C. Nat. Struct. Biol. 2003; 10: 394-401Crossref PubMed Scopus (237) Google Scholar) show similar molecular architectures. The presence of a switch helix has only been observed in the structures of FhuA and FecA but not in FepA and BtuB, implying that this structure element is not essential for TonB recognition in general. The pathway of the ligand from the binding site to the periplasm and the mechanism of its transport have not yet been elucidated. Two possibilities are discussed in the literature: 1) conformational change of the cork domain opens up a channel large enough for the siderophore to slide through (18Ferguson A.D. Deisenhofer J. Biochim. Biophys. Acta. 2002; 1565: 318-332Crossref PubMed Scopus (120) Google Scholar, 10Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar) or 2) the cork domain leaves the barrel together with the bound siderophore (19Usher K.C. Ozkan E. Gardner K.H. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10676-10681Crossref PubMed Scopus (54) Google Scholar). A highly conserved motif among all TonB-dependent siderophore receptors is the TonB-box (residues 7–11: DTITV in FhuA), which plays an important role in the receptor-TonB interaction (20Larsen R.A. Foster-Hartnett D. McIntosh M.A. Postle K. J. Bacteriol. 1997; 179: 3213-3221Crossref PubMed Google Scholar, 21Cadieux N. Kadner R.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10673-10678Crossref PubMed Scopus (145) Google Scholar). The TonB-box is located at the periplasmic side of the cork domain close to the switch helix. Furthermore, the globular domains of FhuA and FepA are exchangeable without loss of substrate specificity. For example, a mixed mutant consisting of an FhuA-barrel and an FepA-cork retains the specificity for ferrichrome, the natural substrate for FhuA (23Scott D.C. Cao Z. Qi Z. Bauler M. Igo J.D. Newton S.M. Klebba P.E. J. Biol. Chem. 2001; 276: 13025-13033Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Different cork-barrel combinations from several bacterial strains led to the same results (24Killmann H. Braun M. Herrmann C. Braun V. J. Bacteriol. 2001; 183: 3476-3487Crossref PubMed Scopus (37) Google Scholar). Complexes between wt FhuA or wt FepA with the periplasmic domain of TonB were characterized in vitro (25Moeck G.S. Letellier L. J. Bacteriol. 2001; 183: 2755-2764Crossref PubMed Scopus (56) Google Scholar). However, up to now there has been no in vitro evidence for interactions between the receptor lacking the cork domain and the TonB protein, and new investigations of FepA indicated that the barrel domain alone could not behave as an active transporter (26Vakharia H.L. Postle K. J. Bacteriol. 2002; 184: 5508-5512Crossref PubMed Scopus (28) Google Scholar). The TonB protein of E. coli is composed of 239 amino acids of which 17% are proline residues. Most of these are located between residues 75 and 107, spanning the periplasmic space to link the outer membrane receptor with the cytoplasmic membrane (27Postle K. Skare J.T. J. Biol. Chem. 1988; 263: 11000-11007Abstract Full Text PDF PubMed Google Scholar). The elongated conformation of this proline-rich region has been demonstrated by NMR studies (28Evans J.S. Levine B.A. Trayer I.P. Dorman C.J. Higgins C.F. FEBS Lett. 1986; 208: 211-216Crossref PubMed Scopus (74) Google Scholar). This region is not essential for the process of energy transduction (29Larsen R.A. Wood G.E. Postle K. Mol. Microbiol. 1994; 12: 857Crossref PubMed Scopus (0) Google Scholar). Two other significant regions can be distinguished: 1) a hydrophobic region at the N terminus (residues 1–32) anchoring TonB to the cytoplasmic membrane. The amino acids between Ser-16 and His-20 were found to be essential for the interaction with the membrane-embedded proteins ExbB and ExbD (30Larsen R.A. Postle K. J. Biol. Chem. 2001; 276: 8111-8117Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and 2) a C-terminal domain that forms the contact to the outer membrane receptor. The three-dimensional structure of a C-terminal fragment (residues 155–239) reveals a cylinder-shaped dimer (1Chang C. Mooser A. Pluckthun A. Wlodawer A. J. Biol. Chem. 2001; 276: 27535-27540Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Each monomer contains three β-strands and a short α-helix arranged in a dimer so that the six β-strands build a large antiparallel β-sheet. The first 10 N-terminal amino acids of this fragment are not visible in the electron density map because of their flexibility. The structure of another energy transducing protein, TolA from Pseudomonas aeruginosa, has been solved recently (31Witty M. Sanz C. Shah A. Grossmann J.G. Mizuguchi K. Perham R.N. Luisi B. EMBO J. 2002; 21: 4207-4218Crossref PubMed Scopus (46) Google Scholar). Despite a sequence identity of only 24% (Lalign server) the crystal structure of the periplasmic domain of TolA shows a similar structure and topology, however without dimer formation. The importance of the dimer formation for the mechanism of energy transduction is thus not yet understood. However, it has been shown that a region of TonB contributing the critical interaction with the receptor is located around amino acid 160 (32Günter K. Braun V. FEBS Lett. 1990; 274: 85-88Crossref PubMed Scopus (70) Google Scholar). This finding was supported by the observation that synthetic nonapeptides with sequence identity to the amino acid region between residues 150 and 166 of TonB are able to inhibit the capacity of wt FhuA to transport siderophores (33Killmann H. Herrmann C. Torun A. Jung G. Braun V. Microbiology. 2002; 148: 3497-3509Crossref PubMed Scopus (30) Google Scholar). To understand the role of the C-terminal domain of TonB in the interaction with FhuA, we have investigated FhuA-TonB interactions using purified C-terminal TonB fragments of different lengths shown in Fig. 1 (consisting of 77, 86, 96, 106, 116, or 126 amino acid residues, respectively). All TonB fragments except TonB-77 were able to form a complex with FhuA. Analytical ultracentrifugation experiments and tryptophan fluorescence measurements also showed that the fragments with 86 or more amino acid residues behave differently than TonB-77. In parallel, we analyzed the ability of these TonB fragments to inhibit ferrichrome (FC) and ferric citrate (Cit) uptake in vivo and to protect cells against attack by bacteriophage Φ80. Construction of Plasmids Encoding TonB Proteins—All constructions, with the exception of pBADTonB118, were created using PCR, and the products were first cloned into an intermediate vector (pSKII+ or pKSII+). The oligodeoxynucleotides used are listed in Table I. The plasmid pCSTonB30 (34Howard S.P. Herrmann C. Stratilo C.W. Braun V. J. Bacteriol. 2001; 183: 5885-5895Crossref PubMed Scopus (38) Google Scholar), which encodes residues 33–239 of the periplasmic domain of TonB cloned into pET30a (Novagen), was used as a template to generate the four smaller tonB fragments. Standard PCR conditions were used, with US10–US12 and US26 being the forward primers unique for each fragment as indicated, each one giving a PstI cut site on the 5′-end of the fragment, and US5 as the return primer, creating a HindIII restriction site on the 3′-end of the fragment. In combination with US5, oligonucleotide US10 was used to create pBADTB86, oligonucleotide US11 for pBADTB77, oligonucleotide US12 for pBADTB96, and oligonucleotide US26 for pBADTB106. Each fragment encodes the final number of amino acids of the periplasmic domain of TonB as specified by the TonB fragment number, i.e. pBADTB77 encodes the final 76 amino acids of the periplasmic TonB domain plus a methionine as the first amino acid. The PstI-HindIII-digested product was then electrophoresed, and the TonB fragment isolated and cloned into PstI-HindIII-digested pBAD/gIII. The construct pBADBTB118 was obtained by digesting pMFTLP (34Howard S.P. Herrmann C. Stratilo C.W. Braun V. J. Bacteriol. 2001; 183: 5885-5895Crossref PubMed Scopus (38) Google Scholar) with PstI and HindIII and cloning the fragment into PstI-HindIII-digested pBAD/gIII. Each of these recombinant clones codes for an 18-amino acid (54-bp) signal sequence provided by the vector. Cloning the TonB fragment into the PstI site of pBAD/gIII downstream of this sequence also adds an 8-amino acid linker at the N-terminal side. For pTB77 to pTB126, UR134, UR135, and UR141 through UR144 were the forward primers for each fragment as indicated, each one creating an NdeI site on the 5′-end of the fragment, and UR136 was the return primer, which hybridizes to the pET30a vector just downstream of the multiple cloning site and contains a Bpu1102I site. Cloning of the resulting PCR fragment back into pCSTonB30 created the plasmids pTB77–pTB126, which in each case expresses the indicated TonB fragment without a signal sequence.Table IOligodeuxynudeotides used in creation of pBADTonB and pTB recombinant clonesOligodeoxy nucleotideSequence (5′ to 3′)US5GAA TTC AAG CTT TTA CCT GTT GAG TAA TAG TCAUS10CTG CAG CAT TAA GCC GTA ATC AGC CUS11CTG CAG CAC CGG CAC GAG CAC AGG CAUS12CTG CAG CAC CGG TTA CCA GTG TGG CTT CAUS26CTG CAG TCA AGT ACA GCA ACG GCT GCA ACC AUR134CAT ATG GCA TTA AGC CGT AAT CAG CCUR135CAT ATG CCG GCA CGA GCA CAG GCAUR136GCT AGT TAT TGC TCA GCG GUR141CAT ATG CCG GTT ACC AGT GTG GCT TCAUR142CAT ATG TCA AGT ACA GCA ACG GCT GCAUR143CAT ATG TTT GAA AAT ACG GCA CCG GCA CUR144CAT ATG AAA CCC GTA GAG TCG CGT C Open table in a new tab Bacterial Strains, Plasmids, and Growth Conditions—The strains and plasmids used in this study are shown in Table II. The media used were Tryptone yeast extract (2xYT), nutrient broth (NB) (Difco) and Luria-Bertani media (LB). The growth temperature was 37 °C for all experiments. Ampicillin was used at a concentration of 100 μg/ml (Ap100). Strain AB2847Δara was created by P1 transduction of leu::Tn10 and Δara714 from LMG194 (Invitrogen) into AB2847 (35Hantke K. Braun V. J. Bacteriol. 1978; 135: 190-197Crossref PubMed Google Scholar).Table IIStrains of E. coli K-12 and plasmids usedStrains and plasmidsGenotypeSourceStrainsAB2847aroB tsx malT thiHantke (35Hantke K. Braun V. J. Bacteriol. 1978; 135: 190-197Crossref PubMed Google Scholar)AB2847ΔaraaroB tsx malT thi Δ ara714 leu::Tn10This studyXL BluerecA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB laclqZDM15::Tn10] StrStrata geneW3110IN(rmD-rmE)1 rph-1Jensen (47Jensen K.F. J. Bacteriol. 1993; 175: 3401-3407Crossref PubMed Scopus (401) Google Scholar)LMG194F-Δ lacX74 gal E thi rpsL Δ phoA (PvuII) Δ ara714 leu::Tn10InvitrogenDH5αΔ (argF lac)U169 endA1 recA1 hadR17 supE44 thil gyrA1 relA1 (F′ φ80 lacZ Δ M15)Hanahan (43Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8213) Google Scholar)PlasmidspCSTonB30tonB fusion in pET30aHoward et al. (34Howard S.P. Herrmann C. Stratilo C.W. Braun V. J. Bacteriol. 2001; 183: 5885-5895Crossref PubMed Scopus (38) Google Scholar)pSK + T/pKS + TColE1 ori, lacZ, AprStratagenepBAD/gIIIpBR322 ori, araBAD promoter, araC, AprInvitrogenpMFTLPfecA′ ′tonB fusion in pMALc2GHoward et al. (34Howard S.P. Herrmann C. Stratilo C.W. Braun V. J. Bacteriol. 2001; 183: 5885-5895Crossref PubMed Scopus (38) Google Scholar)pTB77tonB fragment in pET30aThis studypTB86tonB fragment in pET30aThis studypTB96tonB fragment in pET30aThis studypTB106tonB fragment in pET30aThis studypTB116tonB fragment in pET30aThis studypTB126tonB fragment in pET30aThis studypBADTonB77tonB fragment in pBAD/gIIIThis studypBADTonB86tonB fragment in pBAD/gIIIThis studypBADTonB96tonB fragment in pBAD/gIIIThis studypBADTonB106tonB fragment in pBAD/gIIIThis studypBADTonB119tonB fragment in pBAD/gIIIThis study Open table in a new tab Purification of FhuA—FhuA405.H6 was expressed in E. coli strain AW 740 [ΔompF zcb::Tn10 ΔompC fhuA31] (36Ingham C. Buechner M. Adler J. J. Bacteriol. 1990; 172: 3577-3583Crossref PubMed Google Scholar) on plasmid pHX 405 with a his6 tag inserted between residues 405 and 406 (37Moeck G.S. Bazzaz B.S. Gras M.F. Ravi T.S. Ratcliffe M.J. Coulton J.W. J. Bacteriol. 1994; 176: 4250-4259Crossref PubMed Google Scholar). The protein was purified as described in the literature (38Ferguson A.D. Breed J. Diederichs K. Welte W. Coulton J.W. Protein Sci. 1998; 7: 1636-1638Crossref PubMed Scopus (43) Google Scholar) with the following changes: for binding experiments the purification was stopped before the detergent exchange from LDAO to DDAO. Fractions containing FhuA were concentrated to 10 mg/ml and dialyzed overnight against 50 mm ammonium acetate pH 8.0 with 0.05% LDAO (N,N-dimethydodecylamine-N-oxide/FLUKA). Purification of the C-terminal TonB Fragments—C-terminal fragments of TonB (77, 86, 96, 106, 116 and 126 residues, respectively) were over-expressed in E. coli BL21(DE3) cells containing the plasmids pTB77 to pTB126 (shown in Table II) and induced at OD600 = 0.7 by addition of 0.4 mm IPTG (isopropyl-β-d-thiogalactopyranoside, BioVetra). Protein expression was maintained at 37 °C for 2 h. The pellets from 4 × 500 ml cell culture (2xYT/Kan50) were resuspended in buffer A (20 mm Tris-HCl pH 8.0/100 mm NaCl/1 mm EDTA) and the cells were broken by french press (4000 PSIG/3 passes). After centrifugation at 15,000 g for 30 min the supernatant was loaded on an SP Sepharose cation-exchange column (Amersham Biosciences) and was then washed with buffer A. TonB was eluted from the column with a NaCl gradient at a salt concentration of about 300 mm NaCl. The eluate was then desalted on a Sephadex G25 column (Amersham Biosciences) before loading onto another strong cation-exchange column (Source 15s/Amersham Biosciences). The eluted TonB protein containing about 250 mm NaCl was again desalted on a Sephadex G25 column with buffer A (no EDTA) and yielded protein at a concentration of ca. 4 mg/ml. The mobility of the fragments on 15% SDS-PAGE corresponded to their theoretical molecular masses (Fig. 2). The purification was carried out within 1 day to avoid protein degradation. For analytical ultracentrifugation and crystallization experiments an additional gel filtration step was added. The protein was concentrated up to 10 mg/ml (Amicon spin-column with YMCO 5,000) and glycerol was added to a final concentration of 10%. The TonB sample was then loaded onto a gel filtration column (Superose 12 HR 60/10, Amersham Biosciences). Binding experiments with FhuA were done with TonB fragments that were purified without this gel filtration step but mixed with 0.05% LDAO immediately before the incubation with FhuA. Purification of the FhuA405.H6/TonB Complexes—Protein solutions containing FhuA (10 mg/ml) and TonB fragment (4 mg/ml), respectively, were mixed in a weight ratio of 1:2 resulting in a large molar excess of TonB in the samples. The protein mixture was then incubated overnight in the presence of 60 μm ferrichrome (Mr = 740, Biophore Research). Glycerol was subsequently added to the protein solution to a final concentration of 10%. The sample was then applied to a Superose 12 HR 60/10 column (Amersham Biosciences), equilibrated, and eluted with the following buffer: 20 mm Tris, pH 8.0/50 mm NaCl/0.05% LDAO. The flow rate was kept at 0.1 ml/min. The protein-containing fractions were analyzed by 15% SDS-PAGE and stained with Coomassie blue (Fig. 3). For Western blots to detect TonB we used anti-TonB antiserum from rabbit as previously described (34Howard S.P. Herrmann C. Stratilo C.W. Braun V. J. Bacteriol. 2001; 183: 5885-5895Crossref PubMed Scopus (38) Google Scholar). Crystallization, Data Collection, and Structure Solution—The C-terminal TonB-77 fragment was crystallized under the following conditions: TonB-77 was purified as described above and concentrated to 20 mg/ml (Centricon YM 5,000). Hanging drop crystallization plates were used with 1-ml reservoir solution containing 2.0 m sodium formate and 0.1 m sodium citrate, pH 5.6, mixing 2 μl of reservoir solution with 2 μl of protein solution in the drop. Crystals of the size 120 × 120 × 120 μm3 grew at 18 °C within 2 weeks (Fig. 4). For diffraction data collection single TonB-77 crystals were soaked in cryobuffer: reservoir solution with 20% glycerol for 1 min and were then flash-frozen in liquid nitrogen. X-ray diffraction data were collected at beamline ID14-4 at the Electron Synchrotron Radiation Facility in Grenoble, France. The crystals diffracted to a resolution of 2.5 Å. Raw data were processed with the program package XDS (39Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3240) Google Scholar) to a final resolution of 2.7 Å. Higher resolution shells were omitted from the refinement process because of very high R values (>50%). The space group was determined to be P6422 with the following unit cell parameters: a = 61.58 Å, b = 61.58 Å, c = 121.95 Å, α = 90°, β = 90°, and γ = 120°. The structure of TonB-77 was solved using molecular replacement with the program MOLREP (40Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4173) Google Scholar) and REFMAC5 (41Murshudov G.N. Vagin A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13909) Google Scholar) from the program package CCP4 (42Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19793) Google Scholar). The search model consisted of all protein atoms of the published model of TonB-86 (PDB entry 1IHR). 2The worldwide repository for the processing and distribution of 3-D biological macromolecular structure data. Available at www.rcsb.org/pdb/cgi/explore.cgi?pid=70671060950031&page=0&pdbId=1IHR. Chain tracing and model building was done with the graphical interface O (44Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The program LSQCAB from CCP4 (42Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19793) Google Scholar) was used to calculate the r.m.s.d. for the Cα atoms between TonB-77 and the existing structure 1IHR of TonB-86. Analytical Ultracentrifugation—The purified C-terminal fragments of TonB (77, 86, 96, and 116, respectively) were analyzed by sedimentation velocity and sedimentation equilibrium experiments using an AN 60-Ti rotor 316 in a Beckman XL-A Optima equipped with an optical absorbance system (Ariel Lustig, Biozentrum Basel, Switzerland). All protein solutions were freshly purified and gel-filtrated. The buffer was 20 mm Tris, pH 8.0, and 100 mm NaCl in all experiments. Velocity sedimentation data were obtained from 0.5 mg/ml protein solutions and a rotor speed of 54,000 rpm at room temperature obtaining the sedimentation coefficient (s20,w). Sedimentation equilibrium experiments were done at different concentrations between 0.5 and 2 mg/ml and a rotor speed of 24,000 and 28,000 rpm at room temperature. The partial specific volume (ν) of the proteins was calculated on the basis of the amino acid distribution (45Edelstein S.J. Schachman H.K. Methods Enzymol. 1973; 27: 82-98Crossref PubMed Scopus (52) Google Scholar) and was near the mean value of globular proteins 0.73 cm3/g. These experiments were used to determine the molecular mass (Mr), hydrodynamic radius (RH), and the frictional ratio (f/f0) (46Lebowitz J. Lewis M.S. Schuck P. Protein Sci. 2002; 11: 2067-2079Crossref PubMed Scopus (623) Google Scholar) of the purified TonB fragments. The calculations were done with the computer program SEGAL 3SEGAL program description. Analytical Ultracentrifugation at Biozentrum Basel. Available at www.biozentrum.unibas.ch/personal/jseelig/AUC/software00.html. based on the numerical fitting of the sedimentation equilibrium pattern to one or two exponential functions. Tryptophan Fluorescence of the C-terminal TonB Fragments—Fluorescence spectra were measured from TonB-77, TonB-86, TonB-96, and TonB-116, respectively, at an excitation wavelength of 295 nm over the range from 320 to 400 nm (PerkinElmer Life Sciences, L550B). The fragments were purified as described above and used at a final concentration of 0.1 mg/ml. Assay of Bacteriophage Susceptibility—Susceptibility to bacteriophage φ80λi21 was measured by dropping 5-μl aliquots of 10-fold dilutions of the phage onto freshly poured overlays (100 μl containing ∼108 cells of the various strains added to 3 ml of LB soft agar and poured onto LB plates). The LB soft overlay, LB plates, and bacterial cultures each contained the indicated concentration of arabinose when measuring the effect of the arabinose induction level on the susceptibility of the cells to bacteriophage. The susceptibility was recorded as the –log of the highest dilution of phage that gave a confluent lysis zone of the bacterial lawn. Assay of Siderophore-dependent Growth and Iron Transport—The ability of the strains to gain iron from either ferrichrome (FC) or ferric citrate (Cit) was assayed on NB agar plates (34Howard S.P. Herrmann C. Stratilo C.W. Braun V. J. Bacteriol. 2001; 183: 5885-5895Crossref PubMed Scopus (38) Google Scholar). To limit the free iron available to the cells, dipyridyl was added to both the agar plates and NB soft overlay at a final concentration of 250 μm. When measuring the effect of the arabinose induction level on the ability of the cells to transport iron, the various indicated levels of arabinose were added. Sterile paper discs (6-mm diameter) were saturated with 10 μl of either 1 mm FC, 10 mm sodium citrate, or 100 mm sodium citrate and left to dry. The discs were then placed onto the overlays, which consisted of ∼108 bacteria added to 3 ml of NB soft agar. The plates were i