Sucrose transport through maltoporin mutants of Escherichia coli.
Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.
Van Gelder
P
P
Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
Dutzler
R
R
Dumas
F
F
Koebnik
R
R
Schirmer
T
T
D000409:Alanine / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001692:Biological Transport; D002241:Carbohydrates / Q000737:chemistry; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D018272:Porins / Q000737:chemistry; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics*; D013395:Sucrose / Q000378:metabolism*; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002241:Carbohydrates; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008081:Liposomes; C109237:OmpF protein; D018272:Porins; D011991:Receptors, Virus; C489789:ScrY protein, E coli; C079242:ScrY protein, bacteria; C016532:maltoporins; D013395:Sucrose; D000409:Alanine