Stoichiometry and geometry of the CXC chemokine receptor 4 complex with CXC ligand 12: Molecular modeling and experimental validation

Significance The chemokine receptor axis plays a critical role in numerous physiological and pathological processes, yet the structural basis of receptor interaction with chemokines is poorly understood. Although the community agrees on the existence of two distinct epitopes for recognition of receptors by chemokines, conflicting evidence from structural and mutagenesis studies suggested several possibilities for receptor:chemokine complex stoichiometry. We use a combination of computational, functional, and biophysical approaches to show that despite its dimeric nature, chemokine receptor CXCR4 interacts with its chemokine ligand, CXCL12, in a 1:1 stoichiometry. This result is also likely relevant for other receptor:chemokine pairs. Structural modeling informed by restraints derived from cysteine trapping experiments enabled determination of the receptor:chemokine complex geometry at a medium resolution level. 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.