Sugar transport through maltoporin of Escherichia coli. Role of polar tracks.
The three-dimensional structure of the maltooligosaccharide specific outer membrane channel LamB of Escherichia coli complexed with sugar molecules revealed a hypothetical transport pathway. Sugars are supposed to slide over a stretch of aromatic residues facilitated by continuous making/breaking of hydrogen bonds between the hydroxyl groups of the sugars and charged amino acids, the "polar tracks." The effect of nine single and three multiple mutations in the polar track residues was investigated by current fluctuations, liposome swelling assays, and in vivo uptake of radiolabeled substrates. Additionally, sugar transport through wild-type LamB was investigated by current fluctuation analysis in water and deuterium. This way the effects on k(on) and k(off) could be investigated separately. Analyses of the various mutants revealed a strong effect on the k(on) values. Because steering to the binding site requires only a few interactions, consequently the loss of even one bond will have a strong effect. Deuterium experiments, which changed the characteristic of all hydrogen bonds, showed a strong effect on k(off) rates, because at this stage the sugar has numerous interactions with the channel. Furthermore, all the mutations induces a strong decrease of in vivo uptake of sugars. These results clearly demonstrate the importance of the polar track residues on both on and off rates in sugar transport and reveal a strong cooperative effect of hydrogen bond formation.
Dumas
F
F
Department of Biophysical Chemistry and the Department of Microbiology, Biozentrum, University of Basel, 4056 Basel, Switzerland. fabricedumas@yahoo.fr
Koebnik
R
R
Winterhalter
M
M
Van Gelder
P
P
D001425:Bacterial Outer Membrane Proteins; D001665:Binding Sites; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000493:pharmacokinetics; D003903:Deuterium / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005583:Fourier Analysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D018272:Porins; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014312:Trisaccharides / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D002241:Carbohydrates; D008081:Liposomes; D009844:Oligosaccharides; D018272:Porins; D011991:Receptors, Virus; D014312:Trisaccharides; C016532:maltoporins; C016549:maltohexaose; C008317:maltotriose; D003903:Deuterium