Energetics of the HIV gp120-CD4 binding reaction

Abstract

HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical protein-protein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system.

Figure 1

Analysis of a mixture of CD4 and core gp120 by analytical ultracentrifugation. The main figure shows the sedimentation equilibrium data and the best-fit curve (solid) for a mixture of CD4 and core gp120. Under the fit, the theoretical sum is deconvoluted into contributions from the core gp120-CD4 complex (long dashed line), excess CD4 (short dashed line), and baseline (dashed-dotted line).

Figure 2

CD4-gp120 thermodynamics. Calorimetry data for the titration of WD61 full-length (A) and core (B) gp120 with CD4 in 10 mM Na₂HPO₄, 200 mM NaCl, and 0.5 mM EDTA (pH 7.4). The top panels show raw data in power versus time. The area under each spike is proportional to the heat produced at each injection. The lower panels show integrated areas normalized to the number of moles of CD4 injected at each injection step. Best-fit curves represent binding enthalpy changes of −63 and −62 kcal/mol CD4 for full-length and core gp120, respectively. Equilibrium binding K_D values were determined as 5 nM and 190 nM, respectively.

Figure 3

Temperature dependence of the CD4 binding enthalpy change for WD61 full-length gp120 (open circles) and HXBc2 core gp120 (filled circles). Slopes of the plots, obtained from the best-fit lines shown, yielded the binding heat capacity changes (ΔC°) in Table 2.

Figure 4

Protein-protein binding thermodynamics at 25°C for several classes of protein interactions. Sample numbers here are n = 30, 13, 2, and 2 for protein-protein, antibody-antigen, T cell receptor/MHC peptide, and CD4-gp120 interactions, respectively. Average contributions to the binding free energy from enthalpic and entropic driving forces for protein-protein binding reactions are shown as bars. All values except those for CD4-gp120 were taken from ref. .

Figure 5

Secondary structural changes detected by circular dichroism spectroscopy. CD spectra for a stoichiometric mixture of CD4 (D1D2) with WD61 full-length gp120 at 5 μM shown as difference molar extinction coefficient versus wavelength. Experimental data (solid) are compared with theoretical mixtures computed as the sum of the individual component spectra (dashed). Both full-length and core gp120 proteins exhibited this spectral shift over the temperature range 12–37°C. The shift in spectral intensity to higher wavelengths is consistent with an increase in secondary structure upon binding.

Figure 6

Kinetic analysis of the CD4-gp120 interaction. (A) Normalized binding responses for CD4 (1 μM) over a full-length BH10 gp120 (black), glycosylated HXBc2 core gp120 (red), core-gp120 (blue), and an unmodified sensor surface (gray). Responses were normalized to 100% bound at the start of the dissociation phase. (B) Kinetic data set collected for core gp120 binding to a CD4 surface. Core gp120 was injected over the CD4 surface at concentrations of 975, 325, 108, 36, and 0 nM. The injections were done at a flow rate of 100 μl/min in 10 mM Na₂HPO₄, 200 mM NaCl, and 0.5 mM EDTA (pH 7.4) at 37°C. Each injection was replicated to demonstrate that the binding responses were reproducible. The experimental data (black lines) were globally fit to a single site interaction model (red lines) to determine the binding kinetics.