Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria

Science

Volumn 276, Issue 5316, 1997, Pages 1261-1264

  Jiang, Xunqing ^a   Payne, Marvin A ^a   Cao, Zhenghua ^a   Foster, Samuel B ^a   Feix, Jimmy B ^b   Newton, Salete M C ^a,c   Klebba, Phillip E ^a  

^a UNIVERSITY OF OKLAHOMA   (United States)

^b MEDICAL COLLEGE OF WISCONSIN   (United States)

^c UNIVERSITY OF SÃO PAULO   (Brazil)

Author keywords

[No Author keywords available]

Indexed keywords

PORIN;

ACTIVE TRANSPORT; ARTICLE; CELL MEMBRANE TRANSPORT; CHANNEL GATING; ESCHERICHIA COLI; IRON TRANSPORT; LIGAND BINDING; NONHUMAN; OUTER MEMBRANE; PRIORITY JOURNAL; PROKARYOTE;

BACTERIAL OUTER MEMBRANE PROTEINS; BACTERIAL PROTEINS; BIOLOGICAL TRANSPORT; CARRIER PROTEINS; COLICINS; CYCLIC N-OXIDES; CYSTEINE; ELECTRON SPIN RESONANCE SPECTROSCOPY; ENTEROBACTIN; ESCHERICHIA COLI; ESCHERICHIA COLI PROTEINS; FERRIC COMPOUNDS; INDICATORS AND REAGENTS; ION CHANNEL GATING; LIGANDS; MEMBRANE PROTEINS; MESYLATES; PORINS; PROTEIN CONFORMATION; RECEPTORS, CELL SURFACE; SPIN LABELS;

EID: 0030926805     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5316.1261     Document Type: Article

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34. 2-terminus resides in the cytoplasmic membrane, but downstream regions of its sequence may project across the periplasm and facilitate transport through OM receptors by direct, protein-protein interactions [for review, see R. J. Kadner. Mol. Microbiol. 4, 2027 (1990); K. Postle, ibid., p. 2019; V. Braun, K. Gunter, K. Hantke, Biol. Met. 4, 14 (1991); P. E. Klebba, J. M. Rutz, J. Liu, C. K. Murphy, J. Bioenerg. Biomembr. 25, 603 (1993)].
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39. 12, bacteriophage BF23, and colicin E1 and E3 by BtuB [D. R. DiMasi et al., J. Bacteriol. 115, 56 (1973)]; the recognition of ferric enterobactin and colicins B and D by FepA [S. Guterman, ibid. 114, 1217 (1973); A. P. Pugsley and P. Reeves, ibid. 126, 1052 (1976); R. R. Wayne, K. Frick, J. B. Neilands, ibid., p. 7]; the binding of ferrichrome; bacteriophages T1, T5, and φ80; colicin M; and albomycin to TonA (FhuA) [R. Wayne and J. B. Neilands, ibid. 121, 497 (1975)]; and the binding of colicins A and N to OmpF [H. Benedetti et al., J. Mol. Biol. 217, 429 (1991)].
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41. 12, bacteriophage BF23, and colicin E1 and E3 by BtuB [D. R. DiMasi et al., J. Bacteriol. 115, 56 (1973)]; the recognition of ferric enterobactin and colicins B and D by FepA [S. Guterman, ibid. 114, 1217 (1973); A. P. Pugsley and P. Reeves, ibid. 126, 1052 (1976); R. R. Wayne, K. Frick, J. B. Neilands, ibid., p. 7]; the binding of ferrichrome; bacteriophages T1, T5, and φ80; colicin M; and albomycin to TonA (FhuA) [R. Wayne and J. B. Neilands, ibid. 121, 497 (1975)]; and the binding of colicins A and N to OmpF [H. Benedetti et al., J. Mol. Biol. 217, 429 (1991)].
42. 12, bacteriophage BF23, and colicin E1 and E3 by BtuB [D. R. DiMasi et al., J. Bacteriol. 115, 56 (1973)]; the recognition of ferric enterobactin and colicins B and D by FepA [S. Guterman, ibid. 114, 1217 (1973); A. P. Pugsley and P. Reeves, ibid. 126, 1052 (1976); R. R. Wayne, K. Frick, J. B. Neilands, ibid., p. 7]; the binding of ferrichrome; bacteriophages T1, T5, and φ80; colicin M; and albomycin to TonA (FhuA) [R. Wayne and J. B. Neilands, ibid. 121, 497 (1975)]; and the binding of colicins A and N to OmpF [H. Benedetti et al., J. Mol. Biol. 217, 429 (1991)].
43. The proposed model of FepA folding contains 29 hydrophobic or amphiphilic β strands that create a transmembrane β barrel and 14 surface loops, designated here PL1 through PL14 (8, 9).
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50. Z. T. Farahbakhsh, K. Hideg, W. L. Hubbell, Science 262, 1416 (1993); Y.-K. Shin, C. Levinthal, F. Levinthal, W. L. Hubbell ibid. 259, 960 (1993); W. L. Hubbell and C. Altenbach, Curr. Opin. Struct. Biol. 4, 566 (1994); L. J. Berliner and J. B. Reuben, Spin Labeling: Theory and Applications (Plenum, New York, 1989), vol. 8; C. Altenbach, T. Marti, H. G. Khorana, W. L. Hubbell, Science 248, 1088 (1990); C. Altenbach, S. L Flitsch, H. G. Khorana, W. L. Hubbell, Biochemistry 28, 7806 (1989); Z. T. Farahbakhsh, K. D. Ridge, H. G. Khorana, W. L. Hubbell, ibid. 34, 8812 (1995).
51. FepAE280C contains the site-directed substitution of Cys for Glu at residue 280.
52. At neutrality, MAL6 labels Cys residues about 1000-fold more efficiently than it does Lys; the specificity of MTSL for Cys is essentially absolute [L. J. Berliner, Ann. N.Y. Acad. Sci. 414, 153 (1983)].
53. J. Liu, J. M. Rutz, P. E. Klebba, J. B. Feix, Biochemistry 33, 13274 (1994).
54. The determination of labeling specificity presents an obstacle to the site-directed spin labeling of living cells. In the case of bacteria, however, the selectivity of chemical labeling can be evaluated by the use of null mutants [for example, FepA-deficient (fepA) bacteria; Fig. 1]. Furthermore, bacterial OM proteins contain few Cys residues, and available data suggest that those form disulfide bonds deep in tertiary structure [T. Schirmer, T. A. Keller, Y.-F. Wang, J. P. Rosenbusch, Science 267, 512 (1995); (75)]. Therefore, in combination with site-directed mutagenesis to introduce unique Cys residues, spin labels can be attached and verified at sites of interest within membrane proteins of viable cells.
55. Strongly immobilized spins probably result from steric hindrance of spin-label motion (rotation) by adjoining components of protein structure. Weakly immobilized spins, on the other hand, originate from the localization of the spin label in a less restricted environment, probably in better contact with the aqueous milieu. To determine the percentage of weakly immobilized spins, we double-integrated spectra in region of peaks a and b (3443 to 3477 G), using the Bruker WinEPR analysis software, and calculated the ratio of areas b/(a + b).
56. The instantaneous fraction of weakly immobilized spins was not determined from X-band field sweeps (Fig. 2), because the ferric enterobactin transport reaction occurs much faster (8) than the time required (8 min) to collect the signal average of the six field sweeps shown in Fig. 2. Double integrations of the X-band spectra in Fig. 2 represent an average percentage of weakly or strongly immobilized spins, collected over an 8-min period.
57. X. Jiang and P. E. Klebba, unpublished data.
58. C. S. Klug, W. Su, J. Liu, P. E. Klebba, J. B. Feix, Biochemistry 34, 14230 (1995).
59. MTSL-labeled FepAE280C bound and transported ferric enterobactin comparably to wild-type FepA [S. M. C. Newton and P. E. Klebba, unpublished data; (8, 17)].
60. H. Benedetti, R. Lloubes, C. Lazdunski, L. Letellier, EMBO J. 11, 441 (1992); V. Braun, J. Frenz, K. Hantke, K. Schaller, J. Bacteriol. 142, 162 (1980); D. Baty et al., Biochimie 72, 123 (1990); M. Wisner, D. Freymann, P. Ghosh, R. M. Stroud, Nature 385, 481 (1997). Subsequent to OM penetration, pore-forming bacteriocins like colicin B remain associated with their OM receptors, but traverse the periplasm to form voltage-gated channels in the inner membrane that depolarize cellular energetics.
61. H. Benedetti, R. Lloubes, C. Lazdunski, L. Letellier, EMBO J. 11, 441 (1992); V. Braun, J. Frenz, K. Hantke, K. Schaller, J. Bacteriol. 142, 162 (1980); D. Baty et al., Biochimie 72, 123 (1990); M. Wisner, D. Freymann, P. Ghosh, R. M. Stroud, Nature 385, 481 (1997). Subsequent to OM penetration, pore-forming bacteriocins like colicin B remain associated with their OM receptors, but traverse the periplasm to form voltage-gated channels in the inner membrane that depolarize cellular energetics.
62. H. Benedetti, R. Lloubes, C. Lazdunski, L. Letellier, EMBO J. 11, 441 (1992); V. Braun, J. Frenz, K. Hantke, K. Schaller, J. Bacteriol. 142, 162 (1980); D. Baty et al., Biochimie 72, 123 (1990); M. Wisner, D. Freymann, P. Ghosh, R. M. Stroud, Nature 385, 481 (1997). Subsequent to OM penetration, pore-forming bacteriocins like colicin B remain associated with their OM receptors, but traverse the periplasm to form voltage-gated channels in the inner membrane that depolarize cellular energetics.
63. H. Benedetti, R. Lloubes, C. Lazdunski, L. Letellier, EMBO J. 11, 441 (1992); V. Braun, J. Frenz, K. Hantke, K. Schaller, J. Bacteriol. 142, 162 (1980); D. Baty et al., Biochimie 72, 123 (1990); M. Wisner, D. Freymann, P. Ghosh, R. M. Stroud, Nature 385, 481 (1997). Subsequent to OM penetration, pore-forming bacteriocins like colicin B remain associated with their OM receptors, but traverse the periplasm to form voltage-gated channels in the inner membrane that depolarize cellular energetics.
64. Time-resolved changes in spin-label motion were analyzed and fitted with "Peak Fit" (Jandel Scientific, San Rafael, CA).
65. 10 cells. Thus, even after 20 min in the presence of ferric enterobactin, the bacteria remained saturated with the siderophore.
66. 9 cells (8). In such iron-deficient conditions, the bacteria contained 80,000 to 100,000 FepA proteins per cell (8). The FepA turnover number for ferric enter-obactin, calculated from these data as one molecule per minute per FepA monomer, compares well with the 5-min motion cycle seen in Fig. 5, especially because the transport measurements were made in exponentially growing bacteria. In the spectrometer cavity, the bacteria were rapidly shifted (in 90 s) from a state of dormancy at 4°C to active transport at 37°C.
67. 3+ is itself paramagnetic, and may therefore undergo dipole-dipole interactions with the nitroxide spin labels. Dipolar effects from ferric iron, however, exert a broadening effect on nitroxide spectra, opposite to the results we observed in FepA in vivo. Such dipolar interactions, furthermore, are not expected to manifest either TonB-or energy dependence.
68. D. C. Eichler, M. J. Barber, L. P. Solomonson, Biochemistry 24, 1181 (1985).
69. X. Jiang and P. E. Klebba, data not shown.
70. Bacteria in a 100-μl quartz flat cell were cooled to 4°C or warmed to 37°C in the resonator cavity. The temperatures in the cavity were calibrated and monitored with a thermometer.
71. R. P. Mason, E. B. Giavedoni, A. P. Dalmasso, Biochemistry 16, 1196 (1977).
72. We thank Y. Kotake at the ESR Center of the Oklahoma Medical Research Foundation and E. E. Klebba for helpful discussions and J. Liu, C. Murphy, B. Kalyanaraman, and P. Cook for their comments on the manuscript. Supported by grants from NSF (MCB9212070 and CHE9512996 to P.E.K.) and NIH (GM53836 to P.E.K. and GM51339 to J.B.F.).