Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5

Abstract

Seven-transmembrane segment, G protein-coupled receptors play central roles in a wide range of biological processes, but their characterization has been hindered by the difficulty of obtaining homogeneous preparations of native protein. We have created paramagnetic proteoliposomes containing pure and oriented CCR5, a seven-transmembrane segment protein that serves as the principal coreceptor for human immunodeficiency virus (HIV-1). The CCR5 proteoliposomes bind the HIV-1 gp120 envelope glycoprotein and conformation-dependent antibodies against CCR5. The binding of gp120 was enhanced by a soluble form of the other HIV-1 receptor, CD4, but did not require additional cellular proteins. Paramagnetic proteoliposomes are uniform in size, stable in a broad range of salt concentrations and pH, and can be used in FACS and competition assays typically applied to cells. Integral membrane proteins can be inserted in either orientation into the liposomal membrane. The magnetic properties of these proteoliposomes facilitate rapid buffer exchange useful in multiple applications. As an example, the CCR5-proteoliposomes were used to select CCR5-specific antibodies from a recombinant phage display library. Thus, paramagnetic proteoliposomes should be useful tools in the analysis of membrane protein interactions with extracellular and intracellular ligands, particularly in establishing screens for inhibitors.

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Figure 1: Schematic representation of the formation of paramagnetic CCR5 proteoliposomes.
Figure 2: Protein and lipid composition of CCR5-proteoliposomes.
Figure 3: Confocal microscopy of fluorescently labeled CCR5 proteoliposomes.
Figure 4: Binding of anti-CCR5 antibodies to CCR5 on cells and proteoliposomes.
Figure 5: Ligand-binding properties of CCR5 proteoliposomes.
Figure 6: FACS analysis of antibody binding to CCR5.

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Acknowledgements

We thank Ms. Sheri Farnum and Ms. Yvette McLaughlin for manuscript preparation, Ms. Minou Modabber for artwork, and Ms. Maris Handley for assistance with confocal microscopy. We acknowledge the National Cell Culture Center for supplying the 1D4 antibody. We thank Dr. R. Wyatt, Dr. J. Rhodes, and P. Kolchinsky for providing plasmids and for helpful discussions. This work was supported by grants (AI 41851 and GM 56550) from the National Institutes of Health, by a Center for AIDS Research grant, and by the G. Harold and Leila Mathers Foundation, the Friends 10, William F. McCarty Cooper, and Douglas and Judith Krupp.

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Correspondence to Joseph Sodroski.

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Mirzabekov, T., Kontos, H., Farzan, M. et al. Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5. Nat Biotechnol 18, 649–654 (2000). https://doi.org/10.1038/76501

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