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

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 sys- tematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L cul- ture in a triply isotope-labeled ( 2 H/ 15 N/ 13 C) minimal medium. Thus the material is suitable for NMR spectro- scopic studies. The expected a-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interac- tions of CCR5 with its ligands, RANTES and MIP-1b were assessed by surface plasmon resonance yielding KD values in the nanomolar range. Using size exclusion chromatog- raphy, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oli- gomer 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.