Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes

Abstract

Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane a helices and small ecto-and endodomains. A His 6 -tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-b-D-maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins.

Investigation of Ligand‐Receptor Systems by High‐Resolution Solid‐State NMR: Recent Progress and Perspectives

“…A frequently used strategy for proteoliposome preparation involves saturation of preformed liposomal vesicles with detergents, incubation with the solubilized membrane protein and detergent removal by various methods depending on the physicochemical properties of the detergents [51]. Applications to GPCRs include the functional reconstitution of the N-terminally truncated form of rat NTS-1 [41] and the co-reconstitution of the HIV receptor CCR5 with its coreceptor CD4 [52]. In these studies, the detergent was removed with hydrophobic polystyrene beads.…”

Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy

“…The biochemical and structural characterization of individual transmembrane proteins remains a difficult task given the complex cellular environment containing a large number of other receptors and structural proteins in close proximity. Reconstitution of membrane proteins (MP) by addition of lipids during dialysis for detergent displacement allows sustaining the structure and activity of proteins after detergent‐based purification 1–7. However, the purified protein is now part of a proteoliposome containing multiple molecules that are difficult to define or study due to multiple interactions 8.…”

Cholesterol impacts chemokine CCR5 receptor ligand‐binding activity

“…Reconstitution of functional CCR5 in liposomes, starting from detergent‐solubilized and purified forms and the use of either dialysis or Bio‐beads to remove detergent excess, has been reported . In our work, various procedures were assayed for the reconstitution of the detergent‐solubilized CCR5 in both solid supported lipid bilayers and small unilamellar vesicles (SUVs) including the detergent dilution approach whereby the detergent dilution in the receptor sample is decreased below CMC of the detergent mixture and the use of Bio‐beads to remove excess of detergent.…”