Epitope Mapping of CCR5 Reveals Multiple Conformational States and Distinct but Overlapping Structures Involved in Chemokine and Coreceptor Function

• Published in: The Journal of biological chemistry Vol. 274; no. 14; pp. 9617 - 9626
• Main Authors: Lee, B, Sharron, M, Blanpain, C, Doranz, B J, Vakili, J, Setoh, P, Berg, E, Liu, G, Guy, H R, Durell, S R, Parmentier, M, Chang, C N, Price, K, Tsang, M, Doms, R W
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 02-04-1999
• Subjects: AIDS/HIV; Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Cell Line; Chemokines - metabolism; Computer Simulation; Epitope Mapping; Flow Cytometry; Gene Products, env - metabolism; HIV-1; Humans; Models, Molecular; Molecular Sequence Data; Protein Conformation; Receptors, CCR5 - chemistry; Receptors, CCR5 - immunology; Structure-Activity Relationship; Surface Properties
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.14.9617

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope
specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding,
and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes,
extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain.
N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized
residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172.
We found that ECL2-specific mAbs were more efficient than NH 2 - or MD-antibodies in blocking RANTES or MIP-1β binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively
than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability
to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines
and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for
gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane
fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding
states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in
its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic
determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other
mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.