Real-time Exchange of the Lipid-bound Intermediate and Post-fusion States of the HIV-1 gp41 Ectodomain.

The envelope glycoprotein gp41 of the HIV-1 virus mediates its entry into the host cell. During this process, gp41 undergoes large conformational changes and the energy released in the remodeling events is utilized to overcome the barrier associated with fusing the viral and host membranes. Although the structural intermediates of this fusion process are attractive targets for drug development, no detailed high-resolution structural information or quantitative thermodynamic characterization are available. By measuring the dynamic equilibrium between the lipid-bound intermediate and the post-fusion six-helical bundle (6HB) states of the gp41 ectodomain in the presence of bilayer membrane mimetics, we derived both the reaction kinetics and energies associated with these two states by solution NMR spectroscopy. At equilibrium, an exchange time constant of about 12 seconds at 38 °C is observed, and the post-fusion conformation is energetically more stable than the lipid-bound state by 3.4 kcal mol. The temperature dependence of the kinetics indicates that the folding occurs through a high-energy transition state which may resemble a 5HB structure. The energetics and kinetics of gp41 folding in the context of membrane bilayers provide a molecular basis for an improved understanding of viral membrane fusion.