title: Lysine 319 interacts with both glutamic acid 269 and aspartic acid 240 in the lactose carrier of Escherichia coli.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: J I
authors.initials: JI
authors.lastname: Lee
authors.forename: P P
authors.initials: PP
authors.lastname: Hwang
authors.forename: T H
authors.initials: TH
authors.lastname: Wilson
mesh_terms: 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
chemical_list: 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