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Substrate-induced transmembrane signaling in the cobalamin transporter BtuB

Nature Structural & Molecular Biology volume 10, pages 394–401 (2003)Cite this article

Abstract

The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B12). In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (Kd ∼0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.

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Figure 1: Experimental electron density and the structure of BtuB.
Figure 2: Crystallographic structures of BtuB.
Figure 3: Substrate-induced conformational change in the Ton box.
Figure 4: Experimental determination of bound cyanocobalamin and calcium, and their effects upon BtuB structure
Figure 5: Calcium and cyanocobalamin binding in BtuB
Figure 6: Structural differences between the apo BtuB, Ca2+–BtuB and Ca2+–B12–BtuB structures.

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Acknowledgements

We thank M. Purdy and S. Derevakonda for assistance with data collection; W. Minor for useful suggestions related to data collection and processing; C. Bradbeer and N. Cadieux for useful discussion; and R. Kretsinger, R. Nakamoto and E. Perozo for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health. Synchrotron facilities are supported by the Department of Energy (APS SBC, APS IMCA, NSLS X25), Industrial Macromolecular Crystallography Association (APS IMCA), National Science Foundation (CHESS F1) and National Institutes of Health (NSLS X25, CHESS F1).

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  1. David P. Chimento, Arun K. Mohanty and Michael C. Wiener: These authors contributed equally to the work.

Authors and Affiliations

  1. Department of Microbiology, University of Virginia, Charlottesville, 22908, Virginia, USA

    David P. Chimento & Robert J. Kadner

  2. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, 22908, Virginia, USA

    Arun K. Mohanty & Michael C. Wiener

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  1. David P. Chimento
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Correspondence to Michael C. Wiener.

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Chimento, D., Mohanty, A., Kadner, R. et al. Substrate-induced transmembrane signaling in the cobalamin transporter BtuB. Nat Struct Mol Biol 10, 394–401 (2003). https://doi.org/10.1038/nsb914

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