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Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter

By Gail E. Fanucci, Nathalie Cadieux, Robert J. Kadner and David S. Cafiso

Abstract

BtuB is a TonB-dependent outer-membrane transporter for vitamin B(12) (or cyanocobalamin, CN-Cbl) in Escherichia coli. The binding of CN-Cbl is believed to promote an unfolding or undocking of the Ton box, the conserved N-terminal energy coupling motif at the periplasmic surface of the transporter. This structural change may facilitate the interaction of BtuB with the inner membrane protein TonB. In this work, the effect of the receptor binding fragment of colicin E3 (E3R) on the conformation of the Ton box was examined with site-directed spin labeling. Addition of E3R reverses the undocking of the Ton box that is promoted by CN-Cbl, consistent with a competitive binding between the substrate and the colicin fragment. EPR spectroscopy indicates that the Ton box is in a two-state equilibrium between docked and undocked conformations. In the absence of substrate, the docked conformation is the predominant state; however, the equilibrium can be shifted to the undocked state by the addition of detergents or site-specific proline substitutions. Even when the undocking is induced by detergents or by certain proline mutations, E3R binding shifts the equilibrium to the docked conformation. Thus, two competitive extracellular ligands, CN-Cbl and ER3, transduce opposite conformations of the N-terminal Ton box. Substrate binding stabilizes an undocked conformation, whereas E3R binding stabilizes a docked conformation of the Ton box

Topics: Biological Sciences
Publisher: National Academy of Sciences
Year: 2003
DOI identifier: 10.1073/pnas.1932486100
OAI identifier: oai:pubmedcentral.nih.gov:208766
Provided by: PubMed Central
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