TonB-dependent ligand trapping in the BtuB transporter.
TonB-dependent transporters are β-barrel outer membrane proteins occluded by a plug domain. Upon ligand binding, these transporters extend a periplasmic motif termed the TonB box. The TonB box permits the recruitment of the inner membrane protein complex TonB-ExbB-ExbD, which drives import of ligands in the cell periplasm. It is unknown precisely how the plug domain is moved aside during transport nor have the intermediate states between TonB recruitment and plug domain movement been characterized biochemically. Here we employ nanodiscs, native gel electrophoresis, and scintillation proximity assays to determine the binding kinetics of vitamin B12to BtuB. The results show that ligand-bound BtuB recruits a monomer of TonB (TonB∆1-31), which in turn increases retention of vitamin B12within the transporter. The TonB box and the extracellular residue valine 90 that forms part of the vitamin B12binding site are essential for this event. These results identify a novel step in the TonB-dependent transport process. They show that TonB binding to BtuB trap the ligand, possibly until the ExbB-ExbD complex is activated or recruited to ensure subsequent transport.
Department of Biochemistry & Molecular Biology, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
Allan
A
Mills
Department of Biochemistry & Molecular Biology, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
Hai-Tuong
HT
Le
Department of Biochemistry & Molecular Biology, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Electronic address: fduong@mail.ubc.ca.
Franck
F
Duong
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C057616:tonB protein, Bacteria; D014805:Vitamin B 12
Membrane; Nanodiscs; Scintillation proximity assay; TonB; Transporters; β-barrel proteins