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

• Published in: Journal of molecular biology Vol. 313; no. 5; pp. 1181 - 1193
• Main Authors: Navenot, J M, Wang, Z X, Trent, J O, Murray, J L, Hu, Q X, DeLeeuw, L, Moore, P S, Chang, Y, Peiper, S C
• Format: Journal Article
• Language: English
• Published: England 09.11.2001
• Subjects: Alanine - genetics; Alanine - metabolism; Amino Acid Substitution; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - pharmacology; Binding, Competitive; CCR5 protein; CCR5 Receptor Antagonists; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5 - antagonists & inhibitors; Chemokine CCL5 - metabolism; Chemokines - antagonists & inhibitors; Chemokines - chemistry; Chemokines - metabolism; CHO Cells; Cricetinae; Glycoproteins - metabolism; HIV-1 - metabolism; Human immunodeficiency virus 1; Humans; Ligands; Macrophage Inflammatory Proteins - antagonists & inhibitors; Macrophage Inflammatory Proteins - metabolism; Mice; Models, Molecular; Mutation; Protein Binding - drug effects; Protein Structure, Tertiary; Receptors, CCR5 - chemistry; Receptors, CCR5 - genetics; Receptors, CCR5 - metabolism; Recombinant Fusion Proteins - antagonists & inhibitors; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Signal Transduction; Structure-Activity Relationship; Substrate Specificity; Tumor Cells, Cultured; Viral Envelope Proteins - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2001.5086

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.