title: Structure and function of the glucose PTS transporter from Escherichia coli.
abstract: The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.
authors: Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
authors: Jean-Marc
authors: JM
authors: Jeckelmann
authors: Daniel
authors: D
authors: Harder
authors: Stefania A
authors: SA
authors: Mari
authors: Marcel
authors: M
authors: Meury
authors: Zöhre
authors: Z
authors: Ucurum
authors: Daniel J
authors: DJ
authors: Müller
authors: Bernhard
authors: B
authors: Erni
authors: Dimitrios
authors: D
authors: Fotiadis
mesh_terms: D003461:Crystallography; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D051246:Glucose Transport Proteins, Facilitative / Q000737:chemistry* / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins / Q000737:chemistry; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D051246:Glucose Transport Proteins, Facilitative; D008565:Membrane Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase