Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.
Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha ... (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.
Mesh Terms:
Alanine, Amino Acid Substitution, Animals, Antibodies, Monoclonal, Binding, Competitive, CHO Cells, Chemokine CCL3, Chemokine CCL4, Chemokine CCL5, Chemokines, Cricetinae, Glycoproteins, HIV-1, Humans, Ligands, Macrophage Inflammatory Proteins, Mice, Models, Molecular, Mutation, Protein Binding, Protein Structure, Tertiary, Receptors, CCR5, Recombinant Fusion Proteins, Signal Transduction, Structure-Activity Relationship, Substrate Specificity, Tumor Cells, Cultured, Viral Envelope Proteins
Alanine, Amino Acid Substitution, Animals, Antibodies, Monoclonal, Binding, Competitive, CHO Cells, Chemokine CCL3, Chemokine CCL4, Chemokine CCL5, Chemokines, Cricetinae, Glycoproteins, HIV-1, Humans, Ligands, Macrophage Inflammatory Proteins, Mice, Models, Molecular, Mutation, Protein Binding, Protein Structure, Tertiary, Receptors, CCR5, Recombinant Fusion Proteins, Signal Transduction, Structure-Activity Relationship, Substrate Specificity, Tumor Cells, Cultured, Viral Envelope Proteins
J. Mol. Biol.
Date: Nov. 09, 2001
PubMed ID: 11700073
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