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Journal of Bacteriology, June 2002, p. 2994-2999, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.2994-2999.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Sugar Transport through Maltoporin of Escherichia coli: Role of the Greasy Slide

Patrick Van Gelder,1,2,{dagger} Fabrice Dumas,1,{ddagger} Ingrid Bartoldus,3 Nathalie Saint,2,§ Alexei Prilipov,2,|| Mathias Winterhalter,3,# Yanfei Wang,2,{dagger}{dagger} Ansgar Philippsen,1 Jürg P. Rosenbusch,2 and Tilman Schirmer1*

Division of Structural Biology,1 Division of Microbiology,2 Division of Biophysical Chemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland3

Received 18 July 2000/ Accepted 1 March 2002

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.

* Corresponding author. Mailing address: Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland. Phone: 41-61-267 20 89. Fax: 41-61-267 21 09. E-mail: tilman.schirmer{at}unibas.ch.

{dagger} Present address: Department of Ultrastructure, VUB, B-1640 Sint-Genesius Rode, Belgium.

{ddagger} Present address: IPBS, CNRS, F-31077 Toulouse, France.

§ Present address: UMR 5048 CNRS 554 INSERM, Centre de Biochimie Structurale, Montpellier, France.

|| Present address: Ivanovsky Institute of Virology, 123098 Moscow, Russia.

# Present address: Institut de Pharmacologie et de Biolgie Structurale, CNRS, University of Toulouse, Toulouse, France.

{dagger}{dagger} Present address: Department of Biochemistry, Guangzhou Medical College, Guangzhou 510182, People's Republic of China.

Journal of Bacteriology, June 2002, p. 2994-2999, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.2994-2999.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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