Site-directed disulfide bonding reveals an interaction site between energy-coupling protein TonB and BtuB, the outer membrane cobalamin transporter.
Transport of vitamin B(12) across the outer membrane of Escherichia coli, like that of iron-siderophore complexes, is an active transport process requiring a specific outer membrane transporter BtuB, the proton motive force, and the trans-periplasmic energy coupling protein TonB. Interaction between TonB and two of the TonB-dependent siderophore transporters has been detected previously by formaldehyde crosslinking. Here, site-directed disulfide crosslinking demonstrates contact between a conserved region of BtuB, called the TonB-box, and a portion of TonB, previously implicated as the site of suppressors of TonB-box mutations. The specific pattern of disulfide bonding to alternating residues in the TonB-box allowed deduction of the conformation and parallel orientation of the contact region between these two protein segments. Crosslinking at several positions was increased when BtuB was loaded with substrate, and the crosslinking pattern was altered by the presence of substitutions in BtuB that cause a TonB-uncoupled phenotype. This crosslinking process thus reflects protein interactions that are involved in coupling to active transport.
Cadieux
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N
Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Kadner
R J
RJ
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism / Q000502:physiology; D004220:Disulfides / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016296:Mutagenesis; D011485:Protein Binding; D018000:Receptors, Peptide / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018000:Receptors, Peptide; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D003545:Cysteine; D014805:Vitamin B 12