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

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.T he outer membrane of Gram-negative bacteria such as Escherichia coli contains a family of membrane proteins that actively transport large and scarce nutrients, such as iron siderophores or vitamin B 12 (cyanocobalamin, CN-Cbl) into the periplasmic space. These proteins derive energy for transport from the proton chemical potential across the inner membrane by coupling to the transperiplasmic protein TonB and are therefore termed TonB-dependent. High-resolution structures for the vitamin B 12 transporter, BtuB, have recently been obtained (1). These structures are similar to other TonBdependent transporters, such as FepA, FhuA, and FecA (2-5), and consist of a ␤-barrel formed from 22 antiparallel strands with an N-terminal (core) region that is folded into the lumen of the barrel. However, BtuB, which is the smaller than FepA, FhuA, or FecA, has shorter extracellular loops.The molecular mechanism of TonB-dependent transport is presently not known, but it likely involves a major structural rearrangement or unfolding of the N-terminal core region of the transporter during the transport cycle (6). Site-directed spin labeling (SDSL) is a technique that is particularly sensitive to the changes in tertiary contact made by a spin-labeled side chain (7), and this method indicates that there are significant conformational changes in the Ton box on CN-Cbl binding. The Ton box is a highly conserved region near the N terminus of the transporter that is believed to participate in interactions between TonB and the TonB-dependent transporter. SDSL indicates that the Ton box segment of BtuB (residues 6-12; see ...

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae

“…The mixed bicelles were then dialyzed against 20 mM HEPES (pH 7.0) containing 150 mM NaCl. The dialysis buffer was changed 3 times at 24-h intervals (24,25). The protein-lipid bicelles were stored for 2 weeks at 4°C under nitrogen or at Ϫ80°C.…”

TonB-Dependent Transporters: Regulation, Structure, and Function

“…The most definitive work in this area has been done by Cafiso and colleagues using site-directed spin labelling and electron paramagnetic resonance spectroscopy (EPR) to determine position and mobility of the TonB box. They showed that siderophore binding to BtuB results in an unfolded TonB box (termed disordered by crystallographers), whereas the apo structure exhibits a folded (or ordered) TonB box (28). They also showed that reagents used in protein crystallization can inhibit this transition (29), explaining the highly variable results seen in the crystal structures.…”

Use of a Molecular Decoy to Segregate Transport from Antigenicity in the FrpB Iron Transporter from Neisseria meningitidis

“…Despite intensive structural and biophysical investigation over the last 15 years, however, the precise mechanism of substrate transport through the barrel lumen is unclear. Studies by EPR have shown that substrate binding induces an unfolding of the TonB box residues [14], [15]; this must somehow perturb or displace the plug domain to allow the passage of substrate. To complicate matters further, experiments using fluorophores to label specific cysteine residues in the plug domain have indicated that there are significant differences between different transporters [8], [16], [17].…”