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Comparative Study
. 2005 Aug;79(15):9954-69.
doi: 10.1128/JVI.79.15.9954-9969.2005.

Soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis

Marie Pancera  1 , Jacob Lebowitz, Arne Schön, Ping Zhu, Ernesto Freire, Peter D Kwong, Kenneth H Roux, Joseph Sodroski, Richard Wyatt
Affiliations
Comparative Study

Soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis

Marie Pancera et al. J Virol. 2005 Aug.

Abstract

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, mediates binding to the viral receptors and, along with the transmembrane glycoprotein gp41, is a major target for neutralizing antibodies. We asked whether replacing the gp41 fusion/trimerization domain with a stable trimerization motif might lead to a more stable gp120 trimer that would be amenable to structural and immunologic analysis. To obtain stable gp120 trimers, a heterologous trimerization motif, GCN4, was appended to the C terminus of YU2gp120. Biochemical analysis indicated that the gp120-GCN4 trimers were superior to gp140 molecules in their initial homogeneity, and trilobed structures were observable by electron microscopy. Biophysical analysis of gp120-GCN4 trimers by isothermal titration calorimetry (ITC) and ultracentrifugation analyses indicated that most likely two molecules of soluble CD4 could bind to one gp120-GCN4 trimer. To further examine restricted CD4 stoichiometric binding to the gp120-GCN4 trimers, we generated a low-affinity CD4 binding trimer by introducing a D457V change in the CD4 binding site of each gp120 monomeric subunit. The mutant trimers could definitively bind only one soluble CD4 molecule, as determined by ITC and sedimentation equilibrium centrifugation. These data indicate that there are weak interactions between the gp120 monomeric subunits of the GCN4-stabilized trimers that can be detected by low-affinity ligand sensing. By similar analysis, we also determined that removal of the variable loops V1, V2, and V3 in the context of the gp120-GCN4 proteins allowed the binding of three CD4 molecules per trimer. Interestingly, both the gp120-GCN4 variants displayed a restricted stoichiometry for the CD4-induced antibody 17b of one antibody molecule binding per trimer. This restriction was not evident upon removal of the variable loops V1 and V2 loops, consistent with conformational constraints in the wild-type gp120 trimers and similar to those inherent in the functional Env spike. Thus, the gp120-GCN4 trimers demonstrate several properties that are consistent with some of those anticipated for gp120 in the context of the viral spike.

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Figures

FIG. 1.
FIG. 1.
(A, left) Schematic representation of the HIV-1 viral type 1 membrane fusion protein comprising (i) the gp120 receptor binding domain and (ii) the gp41 oligomerization and fusion domains, the transmembrane anchor, and the cytoplasmic tail. The heptad repeats of gp41 are indicated by crosshatching and labeled HR1 and HR2. (A, right) GCN4 trimerization coiled coil, replacing gp41, was appended at the C terminus of the gp120. Gray ovals, CD4 binding site region; ▪, cleavage site mutated to be cleavage defective. (B.) Variant trimeric gp120 glycoproteins were generated, with deletion of the variable loops and of the C terminus (at aa 492 and 497) as schematically diagrammed. An SDS gel of the ΔV1V2V3gp120 and ΔV1V2V3Δ492gp120-GCN4 glycoproteins is shown. Lane 1, ΔV1V2V3gp120 reduced; lane 2, ΔV1V2V3gp120 nonreduced; lane 3, ΔV1V2V3Δ492gp120-GCN4 reduced; lane 4, YU2ΔV1V2V3Δ492gp120-GCN4 nonreduced.
FIG. 2.
FIG. 2.
Gel filtration profile and blue native gels of gp120-GCN4 glycoproteins. (A) Gel filtration profile of the presumptive glycoproteins: aggregates (1), dimer of trimers (2), trimers (3), dimers (4), and monomers (5). (B) Blue native gel. The different fractions are indicated by arrows. Fraction 2, dimer of trimers; fraction 3, trimers; fraction 4, dimers; fraction 5, monomers. (C) Gel filtration profile of purified gp120-GCN4. (D) Blue native gel of the purified gp120-GCN4 trimeric fraction.
FIG. 3.
FIG. 3.
Size exclusion chromatography with Rayleigh light scattering and absorbance detection of protein peaks. The time elution profile of the presumptive gp120 trimer as detected by light-scattering (solid line) and absorbance (dotted line) measurements is shown. Light-scattering and absorbance detector signals are shown in volts.
FIG. 4.
FIG. 4.
Sedimentation equilibrium analysis. (A) Sedimentation equilibrium concentration profiles of the gp120 monomer construct obtained at three different rotor speeds: 17,000 rpm (inverted triangles); 19,000 rpm (triangles), and 21,000 rpm (circles). All plots depicted were measured at 280 nm. Solid lines show the best fit to the exponential distributions after nonlinear regression global modeling of the data at the three different rotor speeds. Residuals of the fitted lines to the experimental data are displayed in the lower panel with the corresponding symbols listed above. The best-fit RMSD error is 0.011. (B) Sedimentation equilibrium concentration profiles of the gp120 trimer construct obtained at three different rotor speeds: 8,000 rpm (triangles), 9,000 rpm (circles), and 12,000 rpm (inverted triangles). All plots depicted were measured at 280 nm. Solid lines show the best fit to the exponential distributions after nonlinear regression global modeling of the data at the three different rotor speeds. Residuals of the fitted lines to the experimental data are displayed in the lower panel with the corresponding symbols listed above. The best-fit RMSD error is 0.012.
FIG. 5.
FIG. 5.
Isothermal titration calorimetry analyses of the gp120-GCN4 glycoproteins. ITC experiments representing the interactions of YU2gp120-GCN4 with IgGb12 (left) and D1D2-CD4 (right) at 37°C are shown. The top panels represent the raw data as power versus time. The area under each spike is proportional to the heat produced for each injection. The bottom panels represent integrated areas per mole of injected ligand (IgGb12 or D1D2-CD4) as a function of molar ratio. The solid line represents the best nonlinear fit to the experimental data. Enthalpy, ΔH = −11.5 kcal/mol; affinity KD = 19 nM; stoichiometry, n = 0.88 (left); enthalpy, ΔH = −58.7 kcal/mol; affinity KD = 65 nM; stoichiometry, n = 0.64 (right).
FIG. 6.
FIG. 6.
Isothermal titration calorimetry analyses of the D457Vgp120-GCN4 glycoproteins. ITC experiments representing the interactions of D457Vgp120-GCN4 with IgGb12 (left) and D1D2-CD4 (right) at 37°C are shown. The top panels represent the raw data as power versus time. The area under each spike is proportional to the heat produced for each injection. The bottom panels represent integrated areas per mole of injected ligand (IgGb12 or D1D2-CD4) as a function of molar ratio. The solid line represents the best nonlinear fit to the experimental data. Ethalpy, ΔH = −10.7 kcal/mol; affinity KD = 114 nM; stoichiometry, n = 0.94 (left); ethalpy, ΔH = −75.6 kcal/mol; affinity KD = 654 nM; stoichiometry, n = 0.24 (right).
FIG. 7.
FIG. 7.
Summary table of the thermodynamic values at 37°C. (A) Interactions of selected gp120 glycoproteins with D1D2-CD4. (B) Interactions of selected gp120 glycoproteins with IgGb12.
FIG. 8.
FIG. 8.
Sedimentation velocity ultracentrifugation. (A) Overlays of the resolved sedimenting species after the Sedfit software is applied for deconvolution of boundary sedimentation velocity data of free gp120-GCN4 (black), complexes of gp120-GCN4/sCD4 at a 1:1 molar ratio (red), gp120-GCN4/sCD4 at a 1:2 molar ratio (orange), gp120-GCN4/sCD4 at a 1:3 molar ratio (blue), and gp120-GCN4/sCD4 at a 1:4 molar ratio (green). (B) Overlays of the resolved sedimenting species after the Sedfit software is applied for deconvolution of boundary sedimentation velocity data of free D457Vgp120-GCN4 (black), complexes of D457Vgp120-GCN4/sCD4 at a 1:1 molar ratio (red), D457Vgp120-GCN4/sCD4 at a 1:3 molar ratio (blue), and D457Vgp120-GCN4/sCD4 at a 1:5 molar ratio (green). (C) Overlays of the resolved sedimenting species after the Sedfit software is applied for deconvolution of boundary sedimentation velocity data of free ΔV1V2V3gp120-GCN4 (black), complexes of ΔV1V2V3gp120-GCN4/sCD4 at a 1:1 molar ratio (red), ΔV1V2V3gp120-GCN4/sCD4 at a 1:3 molar ratio (blue), and ΔV1V2V3gp120-GCN4/sCD4 at a 1:5 molar ratio (green).
FIG. 9.
FIG. 9.
Antigenicity and functional analysis of the YU2gp120-GCN4 glycoproteins. (A) ELISA analysis of gp120, D457Vgp120, gp120-GCN4, and D457Vgp120-GCN4 derived from YU2 by IgGb12 (closed squares), F105 (open squares), and sCD4 (circles). (B) Western blot analysis of CCR5 binding by gp120-GCN4 (lane 1), gp120-GCN4/D1D2-CD4 (lane 2), and gp120-GCN4/D1D2-CD4/17b (lane 3).
FIG. 10.
FIG. 10.
Electron microscopy and graphic representations of trimeric proteins or complexes protein. Most common forms of gp120-GCN4 (A), other forms of gp120-GCN4 (B), gp120-GCN4/sCD4 complex (C), and gp120-GCN4/b12 Fab complex (D).
FIG. 11.
FIG. 11.
Molecular surface of the core gp120 molecule with the position of the D457V mutant. The molecular surface of the core gp120 molecule is indicated in red, the binding site of CD4 is shown in yellow, and the mutated residue 457V is shown in blue. The orientation is such that the viral membrane is located toward the top of the page and the cellular membrane is located toward the bottom of the page.
FIG. 12.
FIG. 12.
Model of binding of the CD4 ligand to the YU2gp120-GCN4 variant glycoproteins. Binding of sCD4 to the WT gp120-GCN4 proteins (left) with a restriction of two sCD4 per trimer (with the possible exception that under conditions of vast excess, three sCD4 molecules may bind) and one sCD4 to the mutant D457Vgp120-GCN4 proteins (right). The D457V mutant residue is schematically denoted in blue in the CD4 binding site of the mutant trimers. The relative affinities of CD4 to the two trimer variants are indicated by a thick arrow (WT, high affinity) or a thin arrow (mutant, low affinity). The symbol (▪) represents sCD4. The different forms of ovals represent the subunits of the trimeric proteins bound or not bound to sCD4, with the double arrows (top panels) indicating subunit interactions that may oppose the conformational changes associated with ligand binding. Based upon sedimentation analysis, the mutant trimer may be more “open” prior to ligand interaction and is represented in that manner (top right).
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