title: Stoichiometry and geometry of the CXC chemokine receptor 4 complex with CXC ligand 12: molecular modeling and experimental validation.
abstract: Chemokines and their receptors regulate cell migration during development, immune system function, and in inflammatory diseases, making them important therapeutic targets. Nevertheless, the structural basis of receptor:chemokine interaction is poorly understood. Adding to the complexity of the problem is the persistently dimeric behavior of receptors observed in cell-based studies, which in combination with structural and mutagenesis data, suggest several possibilities for receptor:chemokine complex stoichiometry. In this study, a combination of computational, functional, and biophysical approaches was used to elucidate the stoichiometry and geometry of the interaction between the CXC-type chemokine receptor 4 (CXCR4) and its ligand CXCL12. First, relevance and feasibility of a 2:1 stoichiometry hypothesis was probed using functional complementation experiments with multiple pairs of complementary nonfunctional CXCR4 mutants. Next, the importance of dimers of WT CXCR4 was explored using the strategy of dimer dilution, where WT receptor dimerization is disrupted by increasing expression of nonfunctional CXCR4 mutants. The results of these experiments were supportive of a 1:1 stoichiometry, although the latter could not simultaneously reconcile existing structural and mutagenesis data. To resolve the contradiction, cysteine trapping experiments were used to derive residue proximity constraints that enabled construction of a validated 1:1 receptor:chemokine model, consistent with the paradigmatic two-site hypothesis of receptor activation. The observation of a 1:1 stoichiometry is in line with accumulating evidence supporting monomers as minimal functional units of G protein-coupled receptors, and suggests transmission of conformational changes across the dimer interface as the most probable mechanism of altered signaling by receptor heterodimers.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093 ikufareva@ucsd.edu thandel@ucsd.edu.
authors.forename: Irina
authors.initials: I
authors.lastname: Kufareva
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Bryan S
authors.initials: BS
authors.lastname: Stephens
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Lauren G
authors.initials: LG
authors.lastname: Holden
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Ling
authors.initials: L
authors.lastname: Qin
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Chunxia
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kawamura
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Ruben
authors.initials: R
authors.lastname: Abagyan
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093 ikufareva@ucsd.edu thandel@ucsd.edu.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
mesh_terms: D001703:Biophysics; D054377:Chemokine CXCL12 / Q000737:chemistry*; D019295:Computational Biology / Q000379:methods; D019281:Dimerization; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D019718:Receptors, CXCR4 / Q000737:chemistry* / Q000235:genetics
chemical_list: D054377:Chemokine CXCL12; D046912:Multiprotein Complexes; D019718:Receptors, CXCR4
keywords: GPCR dimerization; chemokine receptor; cysteine trapping; functional complementation; molecular docking