Conformational changes of gp120 in epitopes near the CCR5 binding site areinduced by CD4 and a CD4 miniprotein mimetic

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (H IV-1) envelope glycoprotein gp120 has been reported to induce conformationa l rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understan d the mechanism of virus docking and cell fusion, we developed a three-comp onent gp120-CD4-17b optical biosensor assay to visualize the CD4-induced co nformational change of gp120 as seen through envelope binding to a neutrali zing human antibody, 17b, which binds to epitopes overlapping the CCR5 bind ing site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear tra nsformation analyses. Adding soluble CD4 (sCD4) increased the association r ate of full-length JR-FL gp120 by 25-fold. This change is consistent with g reater exposure of the 17b binding epitope on gp120 when CD4 is bound and c orrelates with CD4-induced conformational changes in gp120 leading to highe r affinity binding to coreceptor. A smaller enhancement of 17b binding by s CD4 was observed with a mutant of gp120, Delta JR-FL protein, which lacks V 1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL a nd Delta JR-FL argue that CD4-induced conformational changes in the equilib rium state of gp120 lead both to movement of V1/V2 loops and to conformatio nal rearrangement in the gp120 core structure and that both of these lead t o greater exposure of the coreceptor-binding epitope in gp120. A 17b bindin g enhancement effect on JR-R, also was observed with a 32-amino acid charyb dotoxin miniprotein construct that contains an epitope predicted to mimic t he Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding . Results with this construct argue that CD4-mimicking molecules with surro gate structural elements for the Phe 43/Arg 59 components of CD4 are suffic ient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.