The chemokine stromal cell-derived factor-1 (SDF-1/ CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 ␤-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.Chemokines are a number of small (8 -10 kDa) secreted proteins that direct cell migration in immune systems by activating their receptors expressed on the cell surface (1, 2). The chemokine, stromal cell-derived factor-1 (SDF-1, 2 also known as CXCL12) (3, 4), and its receptor, CXCR4 (5-7), play many essential physiological roles, such as homeostatic regulation of leukocyte traffic, hematopoiesis, and embryonic development (8 -11). The interaction between SDF-1 and CXCR4 also controls cancer metastasis (12, 13), and CXCR4 is a co-receptor for T-tropic strains of human immunodeficiency virus, type 1 (5, 14).The most abundant splice variant of SDF-1 (SDF-1␣) is composed of 68 amino acids, and its NMR (15, 16) and crystal structures (17, 18) demonstrated that SDF-1␣ assumes a typical chemokine fold as follows: an unstructured N terminus (Lys 1 -Tyr 7 ) followed by a long flexible loop (N-loop), a three-stranded anti-parallel ␤-sheet, and an ␣-helix. The mutational analyses revealed that although the SDF-1␣ N terminus is critical for the CXCR4-mediated signaling (15), both the N terminus and the N-loop residues are implicated in the receptor binding (15,18,19). In addition, recent mutational analysis suggested that the residues on the SDF-1␣ ␤-sheet are also important for receptor binding (20).CXCR4, composed of 352 amino acids, belongs to the class A G-protein-coupled receptor (GPCR) family, with the seven transmembrane (TM) helices. Whereas GPCR activation is mediated by the conformation...

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