Thiol cross-linking of cytoplasmic loops in the lactose permease of Escherichia coli.
The N- and C-terminal halves of lactose permease, each with a single-Cys residue in a cytoplasmic loop, were coexpressed, and cross-linking was studied in the absence or presence of ligand. Out of the 68 paired-Cys mutants in cytoplasmic loops IV/V and VIII/IX or X/XI, three pairs in loop IV/V and X/XI, (i) Arg135 --> Cys/Thr338 --> Cys, (ii) Arg134 --> Cys/Val343 --> Cys, and (iii) Arg134 --> Cys/Phe345 --> Cys, form a spontaneous disulfide bond, indicating that loops IV/V and X/XI are in close proximity. In addition, specific paired-Cys residues in loop IV/V (132-138) and loop VIII/IX (282-290) or loop X/XI (335-345) cross-link with iodine and/or the homobifunctional cross-linking agents N, N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimide, and 1, 6-bis(maleimido)hexane. The results demonstrate that loop IV/V is close to both loop VIII/IX and loop X/XI. On the other hand, similar though less extensive cross-linking studies indicate that neither the N terminus nor loop II/III appear to be close to loops VIII/IX or X/XI. The findings suggest that the longer cytoplasmic loops are highly flexible and interact in a largely random fashion. However, although a Cys residue at position 134 in loop IV/V, for example, is able to cross-link with a Cys residue at each position in loop VIII/IX or loop X/XI, Cys residues at other positions in loop IV/V exhibit markedly different cross-linking patterns. Therefore, although the domains appear to be very flexible, the interactions are not completely random, suggesting that there are probably at least some structural constraints that limit the degree of flexibility. In addition, evidence is presented suggesting that ligand binding induces conformational alterations between loop IV/V and loop VIII/IX or X/XI.
Kwaw
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Howard Hughes Medical Institute, Departments of Physiology and of Microbiology & Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, California 90095-1662, USA.
Sun
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J
Kaback
H R
HR
D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D027981:Symporters
D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; C021499:lactose permease; D003545:Cysteine