Structural study of the HIV1 gp41 coat protein

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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 38C 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 to an improved understanding of viral membrane fusion. As applies for HIV-1, entry of SARS-CoV-2 into a host cell is also mediated by a Class I viral fusion protein: Spike. This protein, also referred to as S, is responsible for merging the viral and host cell membranes. Atomic resolution models for both the post-fusion 6-helix bundle (6HB) and the prefusion state of Spike have become available from X-ray and cryo-EM studies. However, a mechanistic understanding of the molecular basis for the intervening structural transition, critical for the design of fusion inhibitors, has remained elusive. Using NMR spectroscopy and other biophysical methods, we demonstrate the presence of alpha-helical, membrane-bound, intermediate states of Spikes heptad repeat (HR1 and HR2) domains that are embedded at the lipid-water interface, while in a slow dynamic equilibrium with the post-fusion 6HB state. These results support a model where the HR domains lower the large energy barrier associated with membrane fusion by destabilizing the host and viral membranes, while 6HB formation actively drives their fusion by forcing physical proximity.