Differential substrate-induced signaling through the TonB-dependent transporter BtuB.
The BtuB transporter mediates high-affinity binding and TonB-dependent active transport of vitamin B12 [cyanocobalamin (CNCbl)] across the outer membrane of Escherichia coli. A characteristic feature of TonB-dependent transporters is the Ton box, a conserved sequence near the N terminus and exposed to the periplasm. Crosslinking to TonB and site-directed spin labeling indicated that the Ton box of BtuB undergoes a substantial conformational transition in response to CNCbl binding, but only slight movement was seen in crystal structures. An in vivo method of detecting substrate-induced changes in the Ton box environment measured reaction of a biotin maleimide derivative with cysteine substitutions through the N-terminal region of BtuB between positions 1 and 31. The degree of maleimide labeling of different residues correlated with their accessibility in the crystal structure. Labeling of many positions was increased strongly when CNCbl was present, consistent with the undocking of this region proposed from spin-labeling analyses. The receptor-binding domain of colicin E3, which binds to BtuB competitively with CNCbl, resulted in decreased labeling. Both substrate-induced transitions occur in and beyond the Ton box and were affected by transport-uncoupling substitutions. Thus, two transport substrates that bind competitively to the extracellular face of BtuB stabilize opposite transitions of the Ton box.
Cadieux
Nathalie
N
Department of Microbiology, University of Virginia, Charlottesville, VA 22908-0734, USA.
Phan
Phu G
PG
Cafiso
David S
DS
Kadner
Robert J
RJ
D029681:Arabidopsis Proteins; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases; D018000:Receptors, Peptide / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; C460109:FASS protein, Arabidopsis; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018000:Receptors, Peptide; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D010749:Phosphoprotein Phosphatases