One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli.
Site-directed mutagenesis was performed with the phosphate starvation-inducible outer membrane porin PhoE of Escherichia coli K-12 to study the molecular basis of its anion selectivity. Lysines 18, 29, 64, and 125 were replaced by glutamic acids, and the properties of the mutant porins were investigated in in vivo and in vitro experiments. Lipid bilayer experiments showed that all these mutations had no influence on the pore structure because PhoE and the mutants had the same single channel conductance in KCl solution. Selectivity measurements revealed that the mutations changed the ionic selectivity of PhoE, but the change was dependent on the location of the lysine. Replacement of Lys18 and Lys29 by glutamic acid had a relatively small influence. The effect of the Lys64 substitution was somewhat larger, and the effect of the replacement of Lys125 resulted in the most drastic change in selectivity and in the loss of the interaction of PhoE with polyphosphate, whereas the replacement of the other lysines had no effect on the polyphosphate interaction behavior. The results are consistent with the assumption that the charge spot in PhoE consists of only 1 lysine per monomer, located in position 125 of the primary sequence and probably close to the pore interior.
Bauer
K
K
Lehrstuhl für Biotechnologie der Universität Würzburg, Federal Republic of Germany.
Struyvé
M
M
Bosch
D
D
Benz
R
R
Tommassen
J
J
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002441:Cefsulodin / Q000378:metabolism; D002509:Cephaloridine / Q000378:metabolism; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism; D008051:Lipid Bilayers; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011122:Polyphosphates / Q000378:metabolism*; D018272:Porins; D011485:Protein Binding
D001425:Bacterial Outer Membrane Proteins; D003062:Codon; D007473:Ion Channels; D008051:Lipid Bilayers; D011122:Polyphosphates; D018272:Porins; D008239:Lysine; D002509:Cephaloridine; D002441:Cefsulodin