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Ligand-Specific Opening of a Gated REPORTS brane (11) and TonB promotes uptake Ligand-Specific Opening of a Gated-Porin through channels in the outer membrane, Channel in the Outer Membrane of TonB-mediated transport reactions in- volve energy transduction between two Living Bacteria distinct bilayers. The outer leaflet of the OM contacts Xunqing Jiang, Marvin A. Payne, Zhenghua Cao, the external environment, and various nu- Samuel B. Foster, Jimmy B. Feix, Salete M. C. Newton, trients, noxious agents, and small molecules Phillip E. Klebba* interact with OM proteins during the initial stages of transport (12). Because ferric en- Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic terobactin binds to the outside of a closed cells. The essential role of iron in metabolism makes its acquisition a determinant of channel, through which it subsequently bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, passes, conformational changes in FepA TonB-dependent outer membrane proteins form energized, gated pores that bind iron surface loops are a fundamental part of chelates (siderophores) and internalize them. The time-resolved operation of the Esch- its suspected transport mechanism. To in- erichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin vestigate this possibility, we reacted ni- resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin troxide spin labels with a genetically en- labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane gineered cysteine residue in a ligand-bind- channel, exhibited energy-dependent structural changes during iron and toxin (colicin) ing surface loop [PL5 (13)] and analyzed transport. These changes were not merely associated with ligand binding, but occurred the live bacteria by electron spin reso- during ligand uptake through the outer membrane bilayer. The results demonstrate by nance (ESR) spectroscopy during trans- a physical method that gated-porin channels open and close during membrane transport port. ESR detects the presence of unpaired in vivo. electrons, and the introduction of a stable nitroxide free radical creates a molecular probe that conveys information about the structure and motional dynamics at Transport through biological membranes ins (6) and antibiotics (7). A prototypical its site of attachment. Site-directed spin is selective and directional, providing a gated porin, Escherichia coli FepA, binds labeling has generated extensive informa- regulated influx of solutes required for me- the siderophore ferric enterobactin in tion about membrane proteins in vitro tabolism and an efflux of molecules that closed surface loops (8) and transports it (14), including determinations of second- function outside the cell. Ligand-gated through an underlying transmembrane ary and tertiary structure and character- membrane channels—which exist in a va- channel (9) into the cell. The latter in- ization of conformational change. Pre- riety of organisms and tissues, from bacte- ternalization reaction requires the input of vious ESR results with the purified mutant ria to the human brain—form specific up- energy and the participation of another protein Glu280 3 Cys280 (E280C) (15), take pathways by binding solutes and mov- protein, TonB (10). Because the TonB labeled with either S-(-oxyl-2,2,5,5- ing them through transmembrane pores NH2-terminus resides in the inner mem- tetramethylpyrroline-3-methyl) methane (1). In the outer membrane [OM (2)] of Gram-negative bacteria, TonB-dependent gated porins transport iron in the form of Fig. 1. Nitroxide labeling ferric siderophores (3). These metal che- of FepAE280C in vivo. (A) lates are too big (750 daltons) to traverse ESR analyses of MTSL- labeled bacteria express- the OM through the open channels of ing FepAE280C. KDF541 general porins (4), and channels large (9) containing pITS449 or enough to accommodate them pose a pFepAE280C was grown threat to bacteria, because they would also in LB medium and sub- allow the entry of detergents, antibiotics, cultured into T medium and other noxious molecules. Gated por- plus appropriate nutri- ins have evolved that solve this nutrition- tional supplements at al dilemma by elaborating ligand-binding 1%. After 16 hours, bacteria were harvested and suspended in surface loops that close their large chan- labeling buffer [50 mM Mops and 60 mM NaCl (pH 7.5)]. Spin nels. The acquisition of iron through li- labels were added (to a final concentration of 20 mM MTSL or 10 mM MAL6) and incubated 2 hours at room temperature with gand-gated porins is a determinant of bac- shaking, and the cells were washed twice with reaction buffer terial pathogenesis (5), and the pores containing 0.05% Tween-20 and once with reaction buffer alone. After the labeling reaction, the viability of themselves are routes for the entry of tox- the bacteria was confirmed by plating on LB agar. For ESR analysis, 1010 cells were suspended in 100 ml of 0.01 M NaHPO4 (pH 6.9) containing 0.4% glucose. X-band spectra of MTSL-labeled KDF541/ 1 X. Jiang, M. A. Payne, Z. Cao, S. B. Foster, P. E. Klebba, pFepAE280C ( top), KDF541/pITS449 (fepA ; center), or KDF541 (fepA; bottom) were recorded on a Department of Chemistry and Biochemistry, University of Bruker EMX spectrometer, operating at 9.8 GHz with a total scan range of 150 G and 100-kHz modulation Oklahoma, Norman, OK 73019, USA. frequency, at 4°C in a quartz flat cell. Peaks a and b show the position of strongly and weakly immobilized J. B. Feix, ESR Center of the Biophysics Research Insti- spin labels, respectively. (B)[125I]-labeled protein A Western blots with anti-MAL6 sera. MAL6 was coupled tute, Medical College of Wisconsin, Milwaukee, WI to bovine serum albumin (BSA) and ovalbumin (OVA). New Zealand White rabbits were immunized with 53226, USA. BSA-MAL6 conjugates [100 g/week for 1 month (29)], and polyclonal serum was collected in week 5 and S. M. C. Newton, Department of Chemistry and Bio- m chemistry, University of Oklahoma, Norman, OK 73019, assayed for antigen specificity by enzyme-linked immunosorbent assay (30) and Western blots against USA, and Departamento de Microbiologia, Universidade BSA, BSA-MAL6, OVA, and OVA-MAL6 (lanes 1 through 4, respectively). Lysates (5 3 107 cells) of de Sa˜ o Paulo, Sa˜ o Paulo, Brazil. KDF541, KDF541/pITS449, and KDF541/pFepAE280C, previously labeled with MAL6, as described *To whom correspondence should be addressed. E-mail: above, were reacted with the anti-MAL6 polyclonal serum (lanes 5 through 7, respectively). Arrows [email protected] indicate the positions of (top to bottom) FepAE280C-MAL6, BSA-MAL6, BSA, and OVA-MAL6. www.sciencemag.org z SCIENCE z VOL. 276 z 23 MAY 1997 1261 thiosulfonate (MTSL) or 4-maleimido- pearance of the mobilized form coincided in FepA, either before or during its uptake. tempo [MAL6 (16)] and studied in deter- with the initiation of siderophore trans- Another TonB-dependent ligand that gent or liposomes (17), suggested that port (23), suggesting that ferric enterobac- penetrates via FepA, colicin B, also al- FepA could be specifically modified and tin triggered the conformational changes tered MTSL mobility during its transport, analyzed in vivo (18). Two findings con- but the effects were opposite to those en- firmed this expectation: Bacteria harbor- gendered by the siderophore. The addition ing pfepAE280C were labeled 30- to 50- of saturating colicin B at 37°C progres- fold more than the fepA host strain KDF541 or KDF541 expressing the fepA1 allele that lacks the E280C mutation (Fig. 1). Furthermore, in Western blots (protein immunoblots) of cell lysates with anti- MAL6 sera, the engineered Cys residue at FepA residue 280 was the major spin- labeled site in the bacteria (Fig. 1). Although the in vivo ESR spectra of MTSL attached to residue 280 closely re- sembled its in vitro spectra (17), marked changes appeared during FepA-mediated transport in live bacteria. MTSL-labeled FepA showed a two-component spectrum in vitro, with the majority of its spins strongly immobilized and a small percent- age (,1%) weakly immobilized (17, 19). FepAE280C-MTSL showed similar spec- tra in vivo at 4°C [3% weakly immobilized (peak b in Fig. 2A)], but an increase in sample temperature to 37°C in the pres- ence of the siderophore shifted a signifi- cant proportion of the spin-label popula- tion [16% in Fig. 2E; (20)] to the weakly immobilized state (Fig. 2). The effects of ferric enterobactin on FepA in vivo were distinct from those previously observed in vitro, and the binding of the siderophore caused a slight further immobilization of MTSL at E280C (17). The mobilization of spins in vivo was so conspicuous that we initially suspected chemical release of MTSL from FepA by ferric enterobactin. Other experiments refuted this possibility though. Recooling of the sample to 4°C reversed the effects, regenerating the properties and proportion of strongly im- mobilized spins attached to E280C (Fig. 2). Secondly, MAL6, which forms a chem- ically stable thioether bond with Cys, pro- duced the same results with FepAE280C Fig. 2. Effect of ferric enterobactin on the mobility of in vivo (21). Double integration of the MTSL attached to FepAE280C in live bacteria, ob- X-band spectra, furthermore, showed that served in signal-averaged X-band spectra. KDF541/ spin labels were neither destroyed nor lost pFepAE280C was grown, spin-labeled, and pre- Fig. 3. Effect of colicin B on the mobility of MTSL pared as described in Fig. 1. Signal-averaged X- attached to FepAE280C in live bacteria, observed during the experiment (Fig. 2). The con- band spectra (six field sweeps collected over an in signal-averaged X-band spectra. KDF541/ version of spins from strong to weak im- 8-min period) of spin-labeled bacteria were recorded pFepAE280C was grown, spin-labeled, and pre- mobilization was comparable to changes at 4°C (A) and again after warming the flat cell to pared as described in Fig.
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