Substrate-induced exposure of an energy-coupling motif of a membrane transporter.
BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli. It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in
Merianos
H J
HJ
Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville 22901, USA.
Cadieux
N
N
Lin
C H
CH
Kadner
R J
RJ
Cafiso
D S
DS
D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D001693:Biological Transport, Active; D017124:Conserved Sequence / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018583:Pliability; D011487:Protein Conformation; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013113:Spin Labels; D014805:Vitamin B 12 / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018000:Receptors, Peptide; D013113:Spin Labels; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D014805:Vitamin B 12