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.chemokine receptor | GPCR dimerization | molecular docking | functional complementation | cysteine trapping T he chemokine receptor CXCR4 regulates cell migration during many developmental processes (1, 2). Along with CCR5, it serves as one of the principal coreceptors for HIV entry into leukocytes (3), and is one of the most important chemokine receptors involved in cancer metastasis (4). Stromal-cell derived factor 1 (SDF-1 or CXCL12) was its only known ligand until recently, when CXCR4 was also shown to bind CXCL14 (5) and extracellular ubiquitin (6). Although structures of CXCR4 (7) and CCR5 (8) have been solved with synthetic antagonists, the structural basis for the interaction of CXCR4 (or any other chemokine receptor) with their natural ligands has yet to be determined. Numerous mutagenesis and NMR studies indicate that receptor:chemokine interactions involve two distinct sites (9-12), which has led to a two-site hypothesis of receptor activation (13). The so-called chemokine recognition site 1 (CRS1) (14) includes the N terminus of the receptor interacting with the globular core of the chemokine, whereas chemokine recognition site 2 (CRS2), located within the tra...

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices

“…One of these CXCR4 residues, W94 2.60 , is highly conserved among chemokine receptors, has been implicated in binding the small molecule antagonist IT1t and vMIP-II (8,9), and the equivalent residue in the chemokine receptor CCR5 (W86 2.60 ) has also been shown to play a role in ligand binding (24). Additional signal initiator residues identified in our screen include Y45 1.39 , Y116 3.32 , and E288 7.39 , all previously reported as binding and/ or signaling determinants in CXCR4 (13,19,22,25). In the CXCR4:CXCL12 complex model, each of these four residues directly contacts or is in close proximity to the distal N terminus of CXCL12 (residues K1 and P2), which is widely recognized as the critical domain of the chemokine that initiates signaling (16,26).…”

“…For example, W94A 2.60 , D97A 2.63 , and E288A 7.39 have been previously shown to reduce receptor expression (12,13,22). Substitutions to proline can impact expression and folding, but many were well tolerated.…”

“…CXCR4 residues involved in chemokine engagement. The interaction of CXCL12 with CXCR4 is known to be mediated by two distinct epitopes (1,8,12,(16)(17)(18)(19)(20)(21)(22). In the first [chemokine recognition site 1 (CRS1)], the unstructured N terminus of CXCR4 interacts with the globular core of the chemokine, specifically with the N loop and the 40s loop of CXCL12.…”

“…3B, blue). These engagement residues include D97 2.63 at the top of helix II and D187 ECL2 in ECL2, both of which have previously been implicated in binding CXCL12 (9,17,22,23). On the other side of the pocket, D262 6.58 toward the top of helix VI (consistent with previous studies, ref.…”

Progress Toward Rationally Designed Small-Molecule Peptide and Peptidomimetic CXCR4 Antagonists

“…With respect to the stoichiometry of the CXCL12:CXCR4 complex, several different alternatives have been envisioned (1:1, 1:2, 2:1, 2:2); however, recent studies by Kufareva et al show that the 1:1 complex is the functional unit [6,74].…”