Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodefi ciency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chem okine binding, gp120 binding, and for inducing conformational changes in En v that lead to membrane fusion, We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epito pes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, wh ile nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In additio n, all MD epitopes involved ECL2, including at least residues Lys-171. and Glu-172, We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1 beta binding. By contrast, N-term inal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surpr isingly, ECL2 mAbs were more potent inhibitors of viral infection than N-te rminal mAbs, Thus, the ability to block virus infection did not correlate w ith the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and sugg est that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformationa l changes in Env that lead to membrane fusion and virus infection. Measurem ents of individual antibody affinities coupled with kinetic analysis of equ ilibrium binding states also suggested that there are multiple conformation al states of CCR5. A previously described mAb, 2D7, was unique in its abili ty to effectively block both chemokine and Env binding as well as corecepto r activity. 2D7 bound to a unique antigenic determinant in the first half o f ECL2 and recognized a far greater proportion of cell surface CCR5 molecul es than the other mAbs examined. Thus, the epitope recognized by 2D7 may re present a particularly attractive target for CCR5 antagonists.