Spatial arrangement of the beta-glucoside transporter from Escherichia coli.
The Escherichia coli BglF protein, a sugar permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyzes concomitant transport and phosphorylation of beta-glucosides across the cytoplasmic membrane. Despite intensive studies of PTS permeases, the mechanism that couples sugar translocation to phosphorylation and the nature of the translocation apparatus are poorly understood. Like many PTS permeases, BglF consists of a transmembrane domain, which in addition to transmembrane helices (TMs) contains a big cytoplasmic loop and two hydrophilic domains, one containing a conserved cysteine that phosphorylates the incoming sugar. We previously reported that the big hydrophilic loop, which connects TM VI to TM VII, contains regions that alternate between facing-in and facing-out states and speculated that it is involved in creating the sugar translocation channel. In the current study we used [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), a membrane-impermeative thiol-specific reagent, to identify sites that are involved in sugar transport. These sites map to the regions that border the big loop. Using cross-linking reagents that penetrate the cell, we could demonstrate spatial proximity between positions at the center of the big loop and the phosphorylation site, suggesting that the two regions come together to execute sugar phosphotransfer. Additionally, positions on opposite ends of the big loop were found to be spatially close. Cys accessibility analyses suggested that the sugar induces a change in this region. Taken together, our results demonstrate that the big loop participates in creating the sugar pathway and explain the observed coupling between translocation of PTS sugars from the periplasm to the cytoplasm and their phosphorylation.
Yagur-Kroll
Sharon
S
Department of Molecular Biology The Hebrew University-Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel.
Ido
Ayelet
A
Amster-Choder
Orna
O
D003432:Cross-Linking Reagents / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D007202:Indicators and Reagents / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*
C079638:BglF protein, E coli; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D005960:Glucosides; D007202:Indicators and Reagents; D008565:Membrane Proteins; D008698:Mesylates; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D011494:Protein Kinases