Both CD4 and b12 bind primarily to the outer domain of gp120, which is remarkably well preserved between unliganded, b12- and CD4-bound states ( and ). Their entropies of interaction, however, are very different, with CD4 inducing a 40–50 kcal mol−1
change, and b12 inducing only a 6 kcal mol−1
. This difference in conformational fixation of gp120 is seen in the divergent atomic-mobility values of the domains ().
b12 and CD4 recognition of gp120
The angles of approach of CD4 and b12 to gp120 are similar, although not precisely the same (). If the gp120 outer domains of b12- and CD4-bound structures were superimposed to orient equivalently b12 and CD4, about one-half of CD4 domain 1, which makes all of the contacts with gp120, is not encompassed by the b12 Fab (despite the volume of the Fab variable domains being twice as large as that of domain 1 of CD4). The projection of CD4 outside of the angle of b12 approach suggests that the parameters that sterically restrict b12 binding to the functional spike are not overly stringent.
The contact surfaces of CD4 and b12 on gp120 have considerable overlap ( and Supplementary Fig. 4
). Most of this overlap is on the outer domain, where the CD4-binding loop is a central focus of binding for both CD4 and b12. CD4 and b12, however, interact with the CD4-binding loop quite differently (). b12 uses all three of its CDR heavy-chain loops to grasp virtually all surface-exposed portions of the loop. In contrast, CD4 only binds to one side of the loop, making anti-parallel hydrogen bonds between CD4- and gp120-main-chain atoms.
The primary difference between b12 and CD4 interactions with gp120 involves the conformationally mobile β20/21. For b12, these interactions are peripheral to the binding surface, with alanine substitution of the primary b12 contact with β20/21 (at b12 residue Asn 56) having little impact on overall b12 binding33
. In contrast, CD4 interactions with β20/21 form an integral part of the binding surface, burying 160 Å2
of surface area and forming a topologically contiguous contact surface with the CD4-binding loop.
To delineate further the differences in binding between b12 and CD4, we characterized binding interactions with an HIV-1 gp120 fragment, termed OD1 (ref. 34
). This fragment comprises residues 252–482 and encompasses the entire outer domain including V3 as well as the β20/21 excursion. Binding of b12 to OD1 showed nearly identical rates of association compared to binding of b12 to HXBc2 core gp120, although the dissociation rate was about 15-fold more rapid (Supplementary Fig. 5
). We were unable to detect binding of CD4 to OD1 (Supplementary Fig. 5
). Because CD4 demonstrated virtually no change in on-rate when tested on conformationally stabilized gp120 molecules, we turned to a dodecameric variant of CD4 (D1D2-Igαtp (ref. 35
)), as avidity from multivalent binding provides an effective means by which to reduce off-rate. We observed that D1D2-Igαtp binds with virtually identical rates of association to both OD1 and core gp120 (Supplementary Fig. 5
The results suggest the following series of molecular interactions for b12 and CD4 binding to gp120 (Supplementary Fig. 6
). Initial contact by CD4 occurs with the structurally invariant outer domain, to a surface constitutively exposed on the envelope spike. In primary isolates, which are generally resistant to neutralization by soluble CD4, this interaction is not stable and CD4 readily ‘falls off’. However, at the cell surface (or with dodecameric CD4), multiple CD4 molecules can bind simultaneously to the viral spike and use avidity to enhance stability. The avidity-enhanced outer domain–CD4 complex provides a receptive contact surface for the bridging sheet. A highly coordinated rearrangement of the inner domain allows for formation of the bridging sheet, which welds CD4 into place.
Contact by b12 occurs at the same constitutively exposed surface initially recognized by CD4. However, b12 is able to latch onto this outer domain surface with high affinity, without additional gp120 conformational change. This absence of conformational constraint allows b12 to bind and neutralize primary isolates that otherwise would be protected by conformational masking. In this manner, b12 uses the functionally conserved initial contact site for CD4 on gp120 to neutralize effectively HIV-1 ().
Structural definition of a conformationally invariant, antibody-accessible portion of the CD4-binding site