Structure and plasticity of the human immunodeficiency virus gp41 fusion domain in lipid micelles and bilayers

Abstract

A thorough understanding of the structure of fusion domains of enveloped viruses in changing lipid environments helps us to formulate mechanistic models on how they might function in mediating viral entry by membrane fusion. We have expressed the N-terminal fusion domain of HIV-1 gp41 as a construct that is water-soluble in the absence of membranes, but that also binds with high affinity to lipid micelles and bilayers in their presence. We have solved the structure and studied the dynamics of this domain bound to dodecylphosphocholine micelles by homo- and heteronuclear NMR spectroscopy. The fusion peptide forms a stable hydrophobic helix from Ile(4) to Ala(14), but is increasingly more disordered and dynamic in a segment of intermediate polarity that stretches from Ala(15) to Ser(23). When bound to lipid bilayers at low concentration, the HIV fusion domain is also largely alpha-helical, as determined by CD and FTIR spectroscopy. However, at higher protein/lipid ratios, the domain is partially converted to form beta-structures in lipid bilayers. Controlled lipid mixing occurs at concentrations that support the alpha-helical, but not the beta-strand conformation.

FIGURE 1

Strategy for expression and purification of a soluble version of the HIV-1 gp41 fusion domain. See text for details.

FIGURE 2

¹⁵N-¹H HSQC spectrum of the HIV-1 gp41 fusion domain bound to DPC micelles at 30°C.

FIGURE 3

Sequence, secondary structure propensities based on dihedral bond angles, and NOE connectivities of the HIV-1 gp41 fusion domain bound to DPC micelles at 30°C. In the rows showing φ- and ψ-dihedral angles, triangles indicate α-helix or 3₁₀-helix, circles indicate random coil, and φ- and ψ-angles of residues marked with stars are consistent with either α-helix or β-structure. In the row showing the χ₁ torsion angle, the sizes of squares indicate whether the experimental values allow for one (large), two (medium), or all three (small) staggered rotamer positions χ₁ = −60, 60, 180. Torsion angle restraints that exclude all three rotamer positions are shown as solid circles. The horizontal bars in the rows marked with d indicate distances between respective protons that have been observed by measurements of the corresponding NOEs.

FIGURE 4

Secondary chemical shifts of HIV-1 gp41 fusion domain bound to DPC micelles at 30°C. (Upper panel) Difference of Hα shifts δ_observed – δ_{random coil}. (Lower panel) Difference of Hβ shifts δ_observed – δ_{random coil}.

FIGURE 5

{¹H}-¹⁵N heteronuclear NOEs of HIV-1 gp41 fusion domain bound to DPC micelles at 30°C.

FIGURE 6

Structural models of HIV-1 gp41 fusion domain bound to DPC micelles at 30°C. (a) Forty lowest energy structures with first 18 residues aligned. (b) Same as panel a, but residues 1–24 are shown. (c) Two different views of closest-to-the-mean structure of residues 1–24 with side chains shown. (d) Same as panel c, but only first 18 residues in an electrostatic surface potential representation. Negative potentials are shown in red and positive potentials are shown in blue. Asterisks mark the positions of the Cα atoms of glycines 3, 5, 10, 13, and 16.

FIGURE 7

CD spectra of HIV-1 gp41 fusion domain in solution, bound to DPC micelles, and bound to lipid bilayers composed of POPC/POPG (4:1). (Solid line) 0.1 mM P23H8 in 5 mM HEPES, 10 mM MES buffer, pH 7.4. (Dash-dotted line) Same with 0.2 M NaCl added. (Dash-dot-dot line) Same with 1 M NaCl added. (Dashed line) 0.1 mM P23H8 bound to 10 mM DPC micelles. (Dotted line) 0.1 mM P23H8 bound to 10 mM POPC:POPG (4:1) bilayers. All spectra were recorded at 25°C.

FIGURE 8

ATR-FTIR spectra of HIV-1 gp41 fusion domain bound to planar-supported lipid bilayers composed of POPC/POPG (4:1) at different protein concentrations. (Solid line) Ten micrograms per milliliter P23H8 added to bilayer. (Dashed line) Twenty micrograms per milliliter P23H8 added to bilayer. (Dotted line) Thirty micrograms per milliliter P23H8 added to bilayer. (Dash-dotted line) Forty micrograms per milliliter P23H8 added to bilayer. All spectra were recorded at room temperature. The spectrum of the pure bilayer recorded before the first protein addition was subtracted from each spectrum.

FIGURE 9

Lipid mixing between labeled and unlabeled liposomes composed of POPC/POPG (4:1) mediated by the HIV-1 gp41 fusion domain. Five (a) or three (b) μM P23H8 was added to 200 μM liposomes at time 0 s. Five μM of the control host peptide H7 (GCGKKKK) induces no lipid mixing (c).