Atomic Details

The X-ray crystal structure of core gp120 in complex with CD4 and a neutralizing antibody permitted one of these conformations to be examined at the atomic level (Fig. 2) [16,17]. The core gp120 construct used for crystallization contained deletions at the gp41-interactive region (at the gp120 N/C termini) as well as tripeptide substitutions for two loop regions. The crystal structure showed that core gp120 has two domains: an "inner" domain containing the N and C termini and a heavily glycosylated "outer" domain containing approximately 15 sites of N-linked glycosylation. Extensions emanating from P-hairpins of these two domains combine to form a four-stranded "bridging sheet" minidomain. This minidomain rests on hydrophobic residues contributed by the outer surfaces of the underlying inner and outer domains; thus, the integrity of the bridging sheet is intimately dependent on the precise alignment of the underlying domains.

The CD4 receptor binds at the nexus of the inner domain, outer domain, and bridging sheet. A total of ~ 1600 A2 of surface is buried in the interaction (~ 800 A2 from both CD4 and gp120), which is in the range typical for protein-protein interactions with nanomolar affinity. The interface itself is unusual. Two large interfacial cavities are present, and the gp120 component is contributed by mostly back-bone interactions from six separate sequence stretches. Thermodynamic studies indicate that gp120 undergoes significant conformational change upon binding to CD4. The gp120 glycoprotein appears to fold around CD4, with a coordinated alignment of the inner and outer domains and a reorganization of the bridging sheet [18].

The neutralizing antibody, 17b, captured in the ternary crystal complex binds to the gp120 bridging sheet, to a surface proximal but distinct from that bound by CD4. Sequence analysis shows that this relatively flat surface is highly conserved between different HIV-1 strains, although it appears to be conformationally masked prior to CD4 interaction.

The site of coreceptor binding overlaps with the 17b epitope. Mutational analysis shows that the coreceptor binding surface includes the bridging sheet and part of a variable surface loop, called the V3 loop [19]. Thus, the ternary structure provides a snapshot of the constant regions bound by both CD4 and coreceptor.

The bridging sheet is roughly 50 A distal from the gp120 N and C termini, which interact with gp41. The manner in which

Figure 2 Atomic structure of the ternary complex of core gp120, CD4, and 17b neutralizing antibody. The N-terminal two domains of CD4 are shown in light gray, and the antigen-binding fragment of 17b in dark gray. For the gp120 core of the HXBc2 isolate, a carbon-alpha (Ca) worm representation is shown with inner domain in black, bridging sheet in light gray and outer domain in gray. The protein proximal pentasaccharide for each N-linked glycan is shown in gray all atom representation. The approximate positions of the V1/V2 and V3 variable loops are shown as semitransparent surfaces. (To aid in orienting the viewer, a small boxed inset is shown which depicts gp120 and CD4 in the context of virus and cell surface, respectively. The orientation of gp120 and CD4 in this insert is related to the larger ribbon/atomic depiction by a 90° rotation about a vertical axis.)

Figure 2 Atomic structure of the ternary complex of core gp120, CD4, and 17b neutralizing antibody. The N-terminal two domains of CD4 are shown in light gray, and the antigen-binding fragment of 17b in dark gray. For the gp120 core of the HXBc2 isolate, a carbon-alpha (Ca) worm representation is shown with inner domain in black, bridging sheet in light gray and outer domain in gray. The protein proximal pentasaccharide for each N-linked glycan is shown in gray all atom representation. The approximate positions of the V1/V2 and V3 variable loops are shown as semitransparent surfaces. (To aid in orienting the viewer, a small boxed inset is shown which depicts gp120 and CD4 in the context of virus and cell surface, respectively. The orientation of gp120 and CD4 in this insert is related to the larger ribbon/atomic depiction by a 90° rotation about a vertical axis.)

a signal from coreceptor binding at the bridging sheet/V3 loop is transmitted to gp41 to trigger the fusion machinery is unclear. What is clear is that a number of intermediate conformational steps occur, differentiated antigenically and by accessibility of various neutralizing ligands. While these intermediate structures are currently under investigation, the final fusion-activated, coiled-coil structure of gp41 has been determined at the atomic level by a number of groups (Fig. 1) [20,21].

Figure 3 Mechanisms of humoral immune evasion. The trimeric structure of gp120 is depicted in the orientation obtained by optimization of quantifiable surface parameters [26]. The orientation of the right most protomer is related to the orientation of Fig. 2 by a ~90° rotation about a horizontal axis. This orientation depicts the trimer from the viewpoint of the target cell membrane. The shading scheme for the core gp120 is the same as in Fig. 2 (black Ca worm, inner domain; light gray Ca worm, bridging sheet; gray Ca worm, outer domain; all atom representation, carbohydrate; and semitransparent surfaces, variable loops). Oligomeric shielding of the inner domain by neighboring protomers is apparent, as is the extensive carbohydrate masking of the outer domain surface. The potential shielding of the CD4 binding site by the V1/V2 variable loop is shown with an arrow. The bridging sheet is not formed until CD4 binds; potential conformational alterations in outer domain and V1/V2 loop are highlighted.

Figure 3 Mechanisms of humoral immune evasion. The trimeric structure of gp120 is depicted in the orientation obtained by optimization of quantifiable surface parameters [26]. The orientation of the right most protomer is related to the orientation of Fig. 2 by a ~90° rotation about a horizontal axis. This orientation depicts the trimer from the viewpoint of the target cell membrane. The shading scheme for the core gp120 is the same as in Fig. 2 (black Ca worm, inner domain; light gray Ca worm, bridging sheet; gray Ca worm, outer domain; all atom representation, carbohydrate; and semitransparent surfaces, variable loops). Oligomeric shielding of the inner domain by neighboring protomers is apparent, as is the extensive carbohydrate masking of the outer domain surface. The potential shielding of the CD4 binding site by the V1/V2 variable loop is shown with an arrow. The bridging sheet is not formed until CD4 binds; potential conformational alterations in outer domain and V1/V2 loop are highlighted.

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