Localization of the substrate-binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli.
The mannitol transporter from Escherichia coli, EII(mtl), belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII(mtl) is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC(mtl)). To localize this binding site, 19 single Trp-containing mutants of EII(mtl) were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IIC(mtl). Combined with the available two-dimensional projection maps of IIC(mtl), it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EII(mtl) becomes phosphorylated at Cys(384) in the cytoplasmic B domain. Stably phosphorylated EII(mtl) mutants were constructed, and FRET experiments showed that the position of mannitol in IIC(mtl) remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Milena
M
Opacić
Erwin P P
EP
Vos
Ben H
BH
Hesp
Jaap
J
Broos
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008353:Mannitol / Q000031:analogs & derivatives / Q000378:metabolism; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli