Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes.
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 alpha helices and small ecto- and endodomains. A His6-tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-beta-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.
Devesa
François
F
Institut de Pharmacologie et de Biologie Structurale; CNRS UMR 5089, Toulouse, France.
Chams
Vida
V
Dinadayala
Premkumar
P
Stella
Alexandre
A
Ragas
Aude
A
Auboiroux
Henri
H
Stegmann
Toon
T
Poquet
Yannick
Y
D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D015153:Blotting, Western; D015704:CD4 Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016466:CHO Cells; D054407:Chemokine CCL4; D006224:Cricetinae; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry*; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D008567:Membranes, Artificial; D011485:Protein Binding / Q000502:physiology; D020673:Protein Renaturation; D019713:Receptors, CCR5 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015728:Receptors, HIV / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015704:CD4 Antigens; D054407:Chemokine CCL4; D015699:HIV Envelope Protein gp120; D008081:Liposomes; D019402:Macrophage Inflammatory Proteins; D008567:Membranes, Artificial; D019713:Receptors, CCR5; D015728:Receptors, HIV; D011993:Recombinant Fusion Proteins