Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease.
Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.
Groningen Biomolecular Sciences and Biotechnology Institute and the Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
B A
BA
van Montfort
G K
GK
Schuurman-Wolters
R H
RH
Duurkens
R
R
Mensen
B
B
Poolman
G T
GT
Robillard
D019943:Amino Acid Substitution; D001665:Binding Sites; D003300:Copper / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003545:Cysteine; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008301:Maleimides / Q000494:pharmacology; D008353:Mannitol / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D008301:Maleimides; D009004:Monosaccharide Transport Proteins; D010618:Phenanthrolines; D011994:Recombinant Proteins; D008353:Mannitol; D003300:Copper; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D003545:Cysteine; C025205:1,10-phenanthroline