Structure and function of the glucose PTS transporter from Escherichia coli

Jeckelmann, Jean-Marc; Harder, Daniel; Mari, Stefania A; Meury, Marcel; Ucurum, Zöhre; Müller, Daniel J; Erni, Bernhard; Fotiadis, Dimitrios (2011). Structure and function of the glucose PTS transporter from Escherichia coli. Journal of structural biology, 176(3), pp. 395-403. San Diego, Calif.: Elsevier 10.1016/j.jsb.2011.09.012

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Jeckelmann, Jean-Marc; Harder, Daniel; Meury, Marcel; Ucurum Fotiadis, Zöhre and Fotiadis, Dimitrios José

ISSN:

1047-8477

Publisher:

Elsevier

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 14:23

Last Modified:

06 Dec 2013 13:29

Publisher DOI:

10.1016/j.jsb.2011.09.012

PubMed ID:

21996078

Web of Science ID:

000297089000015

URI:

https://boris.unibe.ch/id/eprint/8144 (FactScience: 213631)

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