Lysine 319 interacts with both glutamic acid 269 and aspartic acid 240 in the lactose carrier of Escherichia coli.
It is believed that there are several charged amino acid residues in membrane-spanning alpha-helices of the lactose carrier of Escherichia coli. Evidence has previously been presented for two different salt bridges in membrane-spanning regions of the lactose carrier. One of these involves an interaction between Asp-237 and Lys-358; another involves interaction between Asp-240 and Lys-319. Additional studies of Lys-319 suggest that it may interact with Glu-269 as well as Asp-240. A cell containing the LacY gene with the mutation Lys-319-->Asn failed to ferment melibiose and after several days melibiose-positive mutants arose on indicator plates. These revertants showed second site mutations which replaced Asp-240 by neutral amino acids (Val or Gly). In addition, a second site mutation showed Glu-269 changed to Asn. Cells containing the mutation Lys-319-->Leu also failed to ferment melibiose and melibiose-positive revertants showed Asp-240-->Ala and Asp-240-->Tyr as well as Tyr-236-->Phe and His-322-->Arg. Second site revertants were also sought from the mutant Glu-269-->Asn which grew poorly on melibiose minimal plates. Melibiose-positive revertants included the double mutant Gln-269/Asn-319. All of the Glu-269-->Asn mutants were extremely defective in transport. It was concluded that Lys-319 interacts with Glu-269 and Asp-240 probably as salt bridges.
Lee
J I
JI
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Hwang
P P
PP
Wilson
T H
TH
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005838:Genotype; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008239:Lysine; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D027981:Symporters
D029968:Escherichia coli Proteins; D005971:Glutamates; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D027981:Symporters; D001224:Aspartic Acid; D018698:Glutamic Acid; C021499:lactose permease; D008553:Melibiose; D007785:Lactose; D008239:Lysine