Identification of residues in the monocyte chemotactic protein-1 that contact the MCP-1 receptor, CCR2

The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of in flammatory diseases. To identify the regions of MCP-1 that contact its rece ptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrolphobicity, or aromaticity at specific positions. The bindi ng affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfe cted CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribut e less than a factor of 10 to the binding affinity. However, two clusters o f primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 Angstrom hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mu tational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region whic h adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cy steines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the r eceptor, and the accumulation of a number of weak interactions results in t he 35 pM affinity observed for the wild-type (WT) protein. The receptor bin ding site of MCP-1 also is significantly different from the binding sites o f RANTES and IL-8, providing insight into the issue of receptor specificity . It was previously shown that the N-terminus of CCR2 is critical for bindi ng MCP-1 [Monteclaro, F. S., and Charo, I. F;. (1996) J. Biol. Chem. 271, 1 9084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 231 86-90]. Point mutations of six acidic residues in this region of the recept or were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we prop ose a model in which the receptor N-terminus lies along the hydrophobic gro ove in an extended fashion, placing the DYDY motif near the basic cluster i nvolving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.