Dynamic membrane topology of the Escherichia coli beta-glucoside transporter BglF.
The Escherichia coli BglF protein, a permease of the phosphoenolpyruvate-dependent phosphotransferase system, catalyzes transport and phosphorylation of beta-glucosides. In addition, BglF regulates bgl operon expression by controlling the activity of the transcriptional regulator BglG via reversible phosphorylation. BglF is composed of three domains; one is hydrophobic, which presumably forms the sugar translocation channel. We studied the topology of this domain by Cys-replacement mutagenesis and chemical modification by thiol reagents. Most Cys substitutions were well tolerated, as demonstrated by the ability of the mutant proteins to catalyze BglF activities. Our results suggest that the membrane domain contains eight transmembrane helices and an alleged cytoplasmic loop that contains two additional helices. The latter region forms a dynamic structure, as evidenced by the alternation of residues near its ends between faced-in and faced-out states. We suggest that this region, together with the two transmembrane helices encompassing it, forms the sugar translocation channel. BglF periplasmic loops are close to the membrane, the first being a reentrant loop. This is the first systematic topological study carried out with an intact phosphotransferase system permease and the first demonstration of a reentrant loop in this group of proteins.
Yagur-Kroll
Sharon
S
Department of Molecular Biology, The Hebrew University-Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
Amster-Choder
Orna
O
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005960:Glucosides / Q000737:chemistry; D007202:Indicators and Reagents / Q000494:pharmacology; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013997:Time Factors
C079638:BglF protein, E coli; D002241:Carbohydrates; D029968:Escherichia coli Proteins; D005960:Glucosides; D007202:Indicators and Reagents; D008024:Ligands; D008565:Membrane Proteins; D008698:Mesylates; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D010770:Phosphotransferases; D011494:Protein Kinases; D003545:Cysteine