C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.
Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Antoine
A
Picciocchi
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Lina
L
Siaučiūnaiteė-Gaubard
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Isabelle
I
Petit-Hartlein
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Rabia
R
Sadir
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Jean
J
Revilloud
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Lydia
L
Caro
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Michel
M
Vivaudou
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France ; Institut Universitaire de France, Paris, France.
Franck
F
Fieschi
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Christophe
C
Moreau
Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France ; CEA, DSV/IBS, Grenoble, France ; CNRS, IBS, Grenoble, France.
Corinne
C
Vivès
D000818:Animals; D018946:Chemokine CCL5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054377:Chemokine CXCL12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D019713:Receptors, CCR5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
C483024:CCL5 protein, human; C584933:CCR5 protein, human; C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D018946:Chemokine CCL5; D054377:Chemokine CXCL12; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D011994:Recombinant Proteins