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Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

Journal of Biomolecular NMR volume 55, pages 79–95 (2013)Cite this article

Abstract

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (2H/15N/13C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding KD values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.

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Acknowledgments

We thank Prof. A. Arseniev for providing the plasmids pET28F10 and pMT10H10 and Prof. A. Spang for the plasmid pCA528, Dr. Paul Jenö and Suzanne Moes for mass spectrometry analysis as well as Dr. Marcel Blommers, Dr. Lukasz Skora and Prof. Sebastian Hiller for stimulating discussions. This work was supported by the EU FP7 Combined Highly Active Anti-Retroviral Microbicides (CHAARM), SNF Grant 31-109,712 and SystemsX.ch (C-CINA).

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  1. Focal Area Structural Biology and Biophysics, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056, Basel, Switzerland

    Maciej Wiktor, Sébastien Morin, Hans-Jürgen Sass & Stephan Grzesiek

  2. Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Mattenstrasse 26, 4058, Basel, Switzerland

    Fabian Kebbel

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  1. Maciej Wiktor
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  2. Sébastien Morin
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  3. Hans-Jürgen Sass
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Correspondence to Stephan Grzesiek.

Electronic supplementary material

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10858_2012_9688_MOESM1_ESM.pdf

The electronic supplementary material contains DNA sequences of cloned constructs, growth media composition and further results on the characterization of E. coli expressed CCR5 by mass spectrometry, size exclusion chromatography, CD, NMR, and SPR. (PDF 5157 kb)

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Wiktor, M., Morin, S., Sass, HJ. et al. Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor. J Biomol NMR 55, 79–95 (2013). https://doi.org/10.1007/s10858-012-9688-4

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  • DOI: https://doi.org/10.1007/s10858-012-9688-4

Keywords

  • CCR5
  • G protein-coupled receptor
  • Expression in E. coli
  • NMR
  • Homology modeling