Phage display reveals multiple contact sites between FhuA, an outer membrane receptor of Escherichia coli, and TonB.
The ferric hydroxamate uptake receptor FhuA from Escherichia coli transports siderophores across the outer membrane (OM). TonB-ExbB-ExbD transduces energy from the cytoplasmic membrane to the OM by contacts between TonB and OM receptors that contain the Ton box, a consensus sequence near the N terminus. Although the Ton box is a region of known contact between OM receptors and TonB, our biophysical studies established that TonB binds to FhuA through multiple regions of interaction. Panning of phage-displayed random peptide libraries (Ph.D.-12, Ph.D.-C7C) against TonB identified peptide sequences that specifically interact with TonB. Analyses of these sequences using the Receptor Ligand Contacts (RELIC) suite of programs revealed clusters of multiply aligned peptides that mapped to FhuA. These clusters localized to a continuous periplasm-accessible surface: Ton box/switch helix; cork domain/beta1 strand; and periplasmic turn 8. Guided by such matches, synthetic oligonucleotides corresponding to DNA sequences identical to fhuA were fused to malE; peptides corresponding to the above regions were displayed at the N terminus of E.coli maltose-binding protein (MBP). Purified FhuA peptides fused to MBP bound specifically to TonB by ELISA. Furthermore, they competed with ligand-loaded FhuA for binding to TonB. RELIC also identified clusters of multiply aligned peptides corresponding to the Ton box regions in BtuB, FepA, and FecA; to periplasmic turn 8 in BtuB and FecA; and to periplasmic turns 1 and 2 in FepA. These experimental outcomes identify specific molecular contacts made between TonB and OM receptors that extend beyond the well-characterized Ton box.
Carter
David M
DM
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Que., Canada H3A 2B4.
Gagnon
Jean-Nicolas
JN
Damlaj
Moussab
M
Mandava
Suneeta
S
Makowski
Lee
L
Rodi
Diane J
DJ
Pawelek
Peter D
PD
Coulton
James W
JW
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C489181:tonB protein, E coli