Fanucci Gail E, Cadieux Nathalie, Kadner Robert J, Cafiso David S
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Proc Natl Acad Sci U S A. 2003 Sep 30;100(20):11382-7. doi: 10.1073/pnas.1932486100. Epub 2003 Sep 17.
BtuB is a TonB-dependent outer-membrane transporter for vitamin B12 (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.
BtuB是大肠杆菌中一种依赖TonB的维生素B12（或氰钴胺素，CN-Cbl）外膜转运蛋白。据信，CN-Cbl的结合会促进转运蛋白周质表面保守的N端能量偶联基序Ton盒的展开或脱离。这种结构变化可能有助于BtuB与内膜蛋白TonB相互作用。在这项工作中，通过定点自旋标记研究了大肠杆菌素E3的受体结合片段（E3R）对Ton盒构象的影响。加入E3R可逆转由CN-Cbl促进的Ton盒脱离，这与底物和大肠杆菌素片段之间的竞争性结合一致。电子顺磁共振光谱表明，Ton盒处于对接和未对接构象之间的双态平衡。在没有底物的情况下，对接构象是主要状态；然而，加入去污剂或位点特异性脯氨酸取代可使平衡向未对接状态移动。即使去污剂或某些脯氨酸突变诱导了未对接，E3R结合也会使平衡向对接构象移动。因此，两种竞争性细胞外配体CN-Cbl和ER3可转导N端Ton盒的相反构象。底物结合稳定未对接构象，而E3R结合稳定Ton盒的对接构象。
