Sugar transport through maltoporin of Escherichia coli: role of the greasy slide.
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.
Van Gelder
Patrick
P
Division of Structural Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Dumas
Fabrice
F
Bartoldus
Ingrid
I
Saint
Nathalie
N
Prilipov
Alexei
A
Winterhalter
Mathias
M
Wang
Yanfei
Y
Philippsen
Ansgar
A
Rosenbusch
Jürg P
JP
Schirmer
Tilman
T
D000409:Alanine / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D003461:Crystallography; D004926:Escherichia coli / Q000378:metabolism*; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D018272:Porins / Q000378:metabolism*; D011991:Receptors, Virus / Q000737:chemistry / Q000378:metabolism*; D014312:Trisaccharides / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D018272:Porins; D011991:Receptors, Virus; D014312:Trisaccharides; C016532:maltoporins; C016549:maltohexaose; C008317:maltotriose; C008315:maltodextrin; D000409:Alanine