title: Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy.
abstract: The mannitol transporter EII(mtl) from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII(mtl) is functional as a dimer and its membrane-embedded C domain, IIC(mtl), harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC(mtl). At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC(mtl). Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII(mtl) is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC(mtl) structure are involved in the sugar translocation. The structure of EII(mtl), as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII(mtl), the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.
authors.affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, AG Groningen, The Netherlands.
authors.forename: Milena
authors.initials: M
authors.lastname: Opačić
authors.forename: Ben H
authors.initials: BH
authors.lastname: Hesp
authors.forename: Fabrizia
authors.initials: F
authors.lastname: Fusetti
authors.forename: Bauke W
authors.initials: BW
authors.lastname: Dijkstra
authors.forename: Jaap
authors.initials: J
authors.lastname: Broos
mesh_terms: D020816:Amino Acid Motifs; D001693:Biological Transport, Active / Q000502:physiology; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008353:Mannitol / Q000737:chemistry* / Q000378:metabolism; 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; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; C026927:5-fluorotryptophan; D008353:Mannitol; D014364:Tryptophan; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli