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 proteinprotein 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. E ntry of enveloped viruses into cells requires transformation of the protective envelope into a fusion-competent state. In the case of the human immunodeficiency virus (HIV-1), infection is initiated by the selective interaction between the viral exterior envelope glycoprotein, gp120, and receptors on the target cell, CD4, and obligatory chemokine receptors (CCR5 or CXCR4). Accumulating biochemical and structural evidence indicates that, in addition to contributing to viral attachment, CD4 triggers conformational alterations in the HIV envelope that promote recognition of the chemokine receptors and ultimately lead to membrane fusion (1, 2). Evidence of CD4-induced conformational changes includes enhanced protease sensitivity in gp120 variable loops (3) and release of gp120 from virus and virus-infected cells (4, 5), as well as exposure or formation of the chemokine receptor site (1, 2) and of the epitopes for neutralizing antibodies that can block chemokine receptor binding (1, 2, 6, 7). By their nature, these biochemical studies primarily probe the peripheral regions of gp120 and indicate that CD4-induced movement of variable loops occurs.Recently, a structure was solved for a truncated core gp120 protein in a ternary complex with soluble CD4 and a Fab fragment of the neutralizing antibody 17b (8). In this complex, gp120 is organized into an inner and outer domain connected by a bridging sheet. Several elements of this structure appear to depend on association with CD4 for their stabilization. In particular, the extended conformation of the bridging sheet, which constitutes key elements of the chemokine receptorbinding site, is stabilized by direct contacts with CD4. In contrast, the structure of CD4 (D1D2) (9, 10) is virtually unchanged in the presence of gp120.Although the crystallographic data provi...

Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins

“…Notably, the interaction of BMS-806 with gp120 was characterized by a binding enthalpy of Ϫ3.4 Ϯ 0.5 kcal͞mol (1 cal ϭ 4.18 J) and a binding entropy of 22 Ϯ 2 cal͞(K ϫ mol). The thermodynamics of BMS-806 binding to gp120 contrasts with the extremely large enthalpy and entropy changes associated with the binding of sCD4 or even CD4 miniproteins (30). The large negative entropy (approximately Ϫ167 cal͞(K ϫ mol) associated with the binding of soluble CD4 (sCD4) has been related to the significant structuring of gp120 residues that is necessary for the formation of the chemokine receptor-binding site (30).…”

“…The thermodynamics of BMS-806 binding to gp120 contrasts with the extremely large enthalpy and entropy changes associated with the binding of sCD4 or even CD4 miniproteins (30). The large negative entropy (approximately Ϫ167 cal͞(K ϫ mol) associated with the binding of soluble CD4 (sCD4) has been related to the significant structuring of gp120 residues that is necessary for the formation of the chemokine receptor-binding site (30). Apparently, BMS-806 does not induce such a large structuring of gp120; the measured entropy change resembles that of small hydrophobic molecules.…”

“…CD4 binding is thought to lock the conformationally flexible free gp120 glycoprotein into a more rigid state that is competent for chemokine receptor binding (30). BMS-806 binding does not introduce a high degree of order into free gp120, nor does it preclude CD4 from doing so.…”

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

“…Furthermore, sCD4 has been reported to bind to envelope proteins, including IIIB gp120, with K D values of 22 to 35 nM (12,59,88). Another study reported K D values in the range of 2.2 to 16 nM for a range of anti-CD4bs Fab fragments (including Fab b12) to MN gp120 (62).…”

Molecular flexibility can influence the stimulatory ability of receptor–ligand interactions at cell–cell junctions

“…However, there were notable outliers, and these peptides stimulated T cells more efficiently than would be predicted from the measured TCRpMHC half-lives. Krogsgaard et al (26) also found that increases in a second parameter, change in heat capacity upon binding, which is often a measure of changes in conformation (27)(28)(29)(30), correlated with the stimulatory ability of the anomalous ligands. Specifically, when T cell stimulation potency was graphed as a function of the product of half-life (measured using soluble molecules) and change in specific heat capacity upon TCRpMHC binding, a good fit to all of the data were obtained.…”