Oligomerization state and supramolecular structure of the HIV-1 Vpu protein transmembrane segment in phospholipid bilayers

Abstract

HIV-1 Vpu is an 81-residue protein with a single N-terminal transmembrane (TM) helical segment that is involved in the release of new virions from host cell membranes. Vpu and its TM segment form ion channels in phospholipid bilayers, presumably by oligomerization of TM helices into a pore-like structure. We describe measurements that provide new constraints on the oligomerization state and supramolecular structure of residues 1-40 of Vpu (Vpu(1-40)), including analytical ultracentrifugation measurements to investigate oligomerization in detergent micelles, photo-induced crosslinking experiments to investigate oligomerization in bilayers, and solid-state nuclear magnetic resonance measurements to obtain constraints on intermolecular contacts between and orientations of TM helices in bilayers. From these data, we develop molecular models for Vpu TM oligomers. The data indicate that a variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, since oligomers of various sizes have similar intermolecular contacts and orientations, molecular models developed from our data are most likely representative of Vpu TM oligomers that exist in host cell membranes.

Figure 1

(a) Full-length HIV-1 Vpu sequence, with three helical segments underlined. Location of N-terminal, transmembrane helical segment is based on solid-state NMR data of Sharpe et al. Central and C-terminal helical segments are based on data of Opella and coworkers., (b) Vpu_1–40 peptides used in 2D CHHC measurements of intermolecular contacts, shown in Figure 8. Samples contain 1:1 mixtures of peptides with uniform ¹⁵N,¹³C-labeling of six Val (cyan), eight Ile (red), or four Ala (green) residues. (c) Vpu_1–40 peptides used in DIPSHIFT measurements of molecular orientation, shown in Figures 9 and 10. Uniformly labeled residues are indicated in red. (d) Y29T-Vpu_1–40 peptide used in analytical ultracentrifugation and PICUP experiments, shown in Figures 2–7. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

(a) Distributions of sedimentation coefficients c(s) derived from sedimentation velocity data for wild-type Vpu_1–40 at loading concentrations of 52 μM with 40 mM C₈E₅ (red line), 100 μM with 40 mM C₈E₅ (blue line), and 130 μM with 10 mM C₈E₅ (green line). A₂₈₀ is the optical absorbance at 280 nm. (b) Distributions for Y29T-Vpu_1–40 at loading concentrations of 54 μM with 40 mM C₈E₅ (red line), 107 μM with 40 mM C₈E₅ (blue line), and 137 μM with 10 mM C₈E₅ (green line). Data were collected at 50 krpm and 20.0°C and analyzed with SEDFIT software. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Possible distributions of Vpu_1–40 oligomers in C₈E₅ micelles, derived by fitting sedimentation equilibrium data to three discrete species of fixed molecular mass and including mass conservation as a constraint. Vertical scales are absolute absorbances at 280 nm for each species. (a) Distributions of Y29T-Vpu_1–40 dimers, tetramers, and hexamers at 10 mM C₈E₅ and peptide loading concentrations of 50, 110, and 160 μM (black, red, and green bars, respectively). (b) Distributions of wild-type Vpu_1–40 dimers, tetramers, and hexamers at 10 mM C₈E₅ and peptide loading concentrations of 45 and 140 μM (black and red bars, respectively). (c) Distributions of Y29T-Vpu_1–40 monomers, dimers, and tetramers at 40 mM C₈E₅ and peptide loading concentrations of 50, 95, and 140 μM (black, red, and green bars, respectively). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

MALDI-TOF mass spectra of PICUP reaction products after the indicated reaction times. Results are shown for crosslinking experiments on wild-type Vpu_1–40 (a) and Y29T-Vpu_1–40 (b). PICUP was performed with a 0.3% mole fraction of peptides in DOPC/DOPG bilayers.

Figure 5

SDS-PAGE analysis of PICUP reaction products after the indicated reaction times, for wild-type Vpu_1–40. (a) Scanned image of the gel, with molecular weight markers in the far left and far right lanes. The control lane is purified Vpu_1–40 directly dissolved in the gel loading buffer at 50 μM peptide concentration, without exposure to bilayers or crosslinking reagents. (b) Densitometry profiles for lanes 2–5, with fits to Gaussian peaks (dashed lines) that represent the indicated N-mers. (c) Apparent relative abundances of N-mers, determined from the fits in panel (b).

Figure 6

Same as Figure 5, but for Y29T-Vpu_1–40.

Figure 7

2D CHHC spectra of V₆I₈-Vpu_1–40 (a,b) V₆A₄-Vpu_1–40 (c,d) and A₄I₈-Vpu_1–40 (e,f) in DOPC/DOPG bilayers at various mixing times, with 1D slices at color-coded positions. Green polygons in the 2D spectra indicate regions where intermolecular crosspeaks would appear if intermolecular ¹³C-¹³C distances were less than roughly 4 Å. Green arrows in the 1D slices indicate observed intermolecular crosspeaks. 2D CHHC experiments were performed at −50°C.

Figure 8

2D spin diffusion spectra and color-coded 1D slices from experiments on V₆I₈-Vpu_1–40 (a, b, c), V₆A₄-Vpu_1–40 (d, e, f), and A₄I₈-Vpu_1–40 (g, h, i) in DOPC/DOPG bilayers. 2D spectra in panels (a, d, g) were obtained with 400 ms spin diffusion periods. 1D slices from these spectra are in panels (b, e, h), with black arrows indicating intermolecular crosspeaks. 1D slices from spectra with 4 ms spin diffusion periods are in panels (c, f, i). 2D spin diffusion experiments were performed at 30°C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 9

2D ¹³C-¹³C NMR spectra of the three Vpu_1–40 samples used in DIPSHIFT experiments, with 1D slices at dashed lines. These are spectra with τ_LG = 0, corresponding to the first point in the curves in Figure 10. DIPSHIFT experiments were performed at 50°C.

Figure 10

Experimental DIPSHIFT curves for individual C_α sites in Vpu_1–40, extracted from 2D ¹³C-¹³C NMR spectra as in Figure 9. Simulated curves with the indicated values of motionally averaged ¹H_α-¹³C_α dipole–dipole couplings are superimposed on the experimental data points.

Figure 11

Example of a molecular model for a Vpu TM oligomer, constructed as described in the text, both before (a) and after (b) energy minimization. Views are shown perpendicular (left) and parallel (right) to the bilayer normal, for a pentamer with τ = −30° and ρ = 0°. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 12

Evaluation of energy-minimized tetrameric (a, d), pentameric (b, e), and hexameric (c, f) molecular models, for helix tilt angles −45° ≤ τ ≤ 45° and rotation angles 0° ≤ ρ ≤ 345°. In panels (a, b, c), models that are consistent with experimental 2D CHHC and spin diffusion spectra are indicated by red regions in the τ,ρ plane. In panels (d, e, f), the deviation Δ (defined in the main text) between experimental and calculated DIPSHIFT results is plotted as a function of τ and ρ, with minima in Δ colored red.

Figure 13

Models for tetrameric (a, b), pentameric (c, d), and hexameric (e, f) Vpu TM oligomers that simultaneously satisfy constraints from 2D CHHC and spin diffusion spectra and DIPSHIFT measurements. These models correspond to τ and ρ values indicated by white arrows in Figure 12. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 14

Comparison of the Vpu TM oligomer model from Figure 13d (a) with Protein Data Bank file 1PI7, as developed by Opella and coworkers (b). For both models, the backbone of residues 7–25 is shown as a ribbon representation, with a ball-and-stick representation of I17, W22, and S23. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]