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Journal of Bacteriology, March 2005, p. 1732-1739, Vol. 187, No. 5
0021-9193/05/$08.00+0     doi:10.1128/JB.187.5.1732-1739.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Multiple Extracellular Loops Contribute to Substrate Binding and Transport by the Escherichia coli Cobalamin Transporter BtuB

Cynthia A. Fuller-Schaefer^1, and Robert J. Kadner^1*

Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia¹

Received 27 August 2004/ Accepted 17 November 2004

The Escherichia coli outer membrane TonB-dependent transporters for iron complexes and cobalamins recognize their multiple and diverse substrates with high specificity and affinity. The X-ray crystallographic structures of several transporters show that the substrate-binding surfaces are comprised of residues from the internal globular domain and multiple extracellular loops. The extracellular loops on the N-terminal half of the transmembrane beta-barrel of the cobalamin transporter BtuB participate in binding of the cofactor calcium atoms and undergo substantial conformation changes upon substrate binding. The functional relevance of the five C-terminal loops was examined by examining the effects of short in-frame deletions. Each loop contributed in different ways to the binding of BtuB substrates. Deletions in loops 7, 8, 9, and 11 strongly decreased cobalamin binding and transport, whereas deletions in loops 8, 9, and 10 affected binding and entry of phage BF23. None of the loops were essential for the action of colicin E1 or E3, which is consistent with the crystallographic observation that the colicin E3 receptor-binding domain can contact almost all of the loops. A deletion in loop 9 or 11 eliminated the ability of cobalamin to inhibit the action of colicin E1. These phenotypes show that there are multiple independent binding elements and point out similarities and differences in binding properties among the TonB-dependent transporters.

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* Corresponding author. Mailing address: Department of Microbiology, University of Virginia, Charlottesville, VA 22908-0734. Phone: (434) 924-2532. Fax: (434) 982-1071. E-mail: rjk{at}virginia.edu.

Present address: Northern Plains Agricultural Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Sidney, MT 59270.

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Journal of Bacteriology, March 2005, p. 1732-1739, Vol. 187, No. 5
0021-9193/05/$08.00+0     doi:10.1128/JB.187.5.1732-1739.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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