Large-scale expression and purification of the major HIV-1 coreceptor CCR5 and characterization of its interaction with RANTES.
The G protein-coupled receptor CCR5 is a human chemokine receptor involved in the activation and migration of leukocytes. CCR5 is also the major HIV-1 coreceptor that, together with human CD4 and the viral glycoprotein gp120, promotes virus entry into host cells. Thus inhibition of the CCR5-gp120 interaction presents a promising route to prevent HIV infections. Atomic structural details of the interaction between CCR5 and its cognate chemokines or gp120 are presently unknown due to the general difficulties of membrane protein structure determination. Here, we report the high-yield expression of human CCR5 in baculovirus-infected Sf9 insect cells. Highly purified (>90%) CCR5 is obtained in detergent-solubilized form at yields of about 1mg/l cell culture. The conformational integrity of recombinant CCR5 after purification is shown by immunoprecipitation with the conformation-dependent monoclonal antibody 2D7, CD and NMR spectroscopy. The detergent micelles contain CCR5 in monomeric and dimeric forms, which can be separated by size exclusion chromatography and characterized individually. Further functional characterization by isothermal titration calorimetry indicates that the recombinant receptor interacts with its cognate chemokine RANTES. This interaction is strongly suppressed when sulfation of CCR5 is inhibited in the insect cells.
Nisius
Lydia
L
Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Rogowski
Marco
M
Vangelista
Luca
L
Grzesiek
Stephan
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000737:chemistry*; D019281:Dimerization; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008823:Micelles; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D019713:Receptors, CCR5 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D018946:Chemokine CCL5; D008024:Ligands; D008823:Micelles; D019713:Receptors, CCR5; D011994:Recombinant Proteins