Identification of Residues in the Monocyte Chemotactic Protein-1 That Contact the MCP-1 Receptor, CCR2

作者：Stefan Hemmerich, Chad D. Paavola, A. Joseph Bloom, Sunil Bhakta, Richard Freedman, Dorit Grunberger, John L. Krstenansky, Simon J. Craddock Lee, Debbie McCarley, Mary A. Mulkins, Belinda Wong, JoeH.B. Pease, Laura S. Mizoue, Tara Mirzadegan, Irene Polsky, Kelly Thompson, Tracy M. Handel, Kurt Jarnagin · 发表于：Biochemistry · 年份：1999 · DOI：10.1021/bi991029m · 被引用次数：143 · 研究领域：Chemokine receptors and signaling、T-cell and B-cell Immunology、Immunotherapy and Immune Responses

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 inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which 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 cysteines 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 receptor,...