Comparative analysis of membrane-associated fusion peptide secondary structure and lipid mixing function of HIV gp41 constructs that model the early pre-hairpin intermediate and final hairpin conformations

Abstract

Fusion between viral and host cell membranes is the initial step of human immunodeficiency virus infection and is mediated by the gp41 protein, which is embedded in the viral membrane. The approximately 20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle structure. The large "N70" (gp41 1-70) and "FP-Hairpin" constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter "FP34" (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final six-helix bundle structure. FP secondary structure at Leu7 of the membrane-associated constructs was probed by solid-state nuclear magnetic resonance and showed populations of molecules with either beta-sheet or helical structure with greater beta-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesterol-dependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for non-fusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.

Fig. 1

(A–C) HIV fusion model. (B–C) Gp120 is not shown in order to focus on gp41 organization. The FP region is indicated using arrows.

Fig. 2

(A) Schematic of the HIV-1 gp41 ectodomain. Primary functional regions are designated by colored boxes and additional functional regions are specified above in braces. Above and below in brackets are the gp41 fragments under study. (B–E) Structural representation of FP34, N70, FP-Hairpin (N70(L6)C39), and Hairpin (N47(L6)C39), respectively, in aqueous solution, with primary sequence below each, color coded to match functional regions in (A). FP34 is shown as a monomer but likely aggregates non-specifically in aqueous solution. The alignment of the NHR and CHR sequences approximately reflects their alignment in crystal structures of NHR and CHR fragment peptides.

Fig. 3

DSC thermograms of Buffer-solubilized (A) 100 μM Hairpin and (B) 80 μM FP-Hairpin. Traces 1 and 3 represent the first and second heating scans and trace 2 is the intervening cooling scan.

Fig. 4

SSNMR REDOR difference spectra representing filtered (A) Ile-4 (B) Leu-7, or (C) Ala-14 ¹³CO signal for FP34 in PC:PG:chol membranes. The FP34:lipid mol ratio was (A,C) 1:40 or (B) 1:25. Each spectrum was processed with 200 Hz Gaussian line broadening and baseline correction, and represents the sum of (A) 78,016 (B) 57,136 or (C) 134,384 S₀ − S₁ scans.

Fig. 5

REDOR difference spectra representing filtered Leu-7 ¹³CO signal for (A1) N70, (A2) FP34, or (A3) FP-Hairpin in PC:PG:chol membranes and (A4) lipid mixing induced between PC:PG:chol vesicles. (B1–B4) are identical to (A1-A4), except that the membrane composition is PC:PG without cholesterol. The protein:lipid mol ratios in the NMR samples were (A1) 1:40 (A2) 1:40 (A3) 1:40 (B1) 1:55 (B2) 1:25 or (B3) 1:30. Each spectrum was processed with 200 Hz Gaussian line broadening and baseline correction and represents the sum of (A1) 76,832 (A2) 200,000 (A3) 97,669 (B1) 144,176 (B2) 51,152 or (B3) 76,128 S₀ − S₁ scans. Dashed lines are at the peak chemical shifts and are assigned to β-sheet or helical conformation. In the lipid mixing assay, time = 0 corresponds to addition of the protein solution to the vesicle solution, the dead time was ~5 seconds, and the protein:lipid mol ratios were N70 or FP34, 1:50, or FP-Hairpin, 1:33.

Fig. 6

REDOR difference spectra representing filtered Leu-7 ¹³CO signal for N70 (A panels) or FP-Hairpin (B panels) in (A1, B1) PC:PG:chol or (A2, B2) PC:PG membranes. The protein:lipid mol ratios are given for each panel and the vertical dashed lines are at the peak chemical shifts that are assigned to β-sheet or helical conformation. Each REDOR spectrum represents the sum of (A1a) 107,472 (A1b) 76,832 (A2a) 144,176 (A2b) 54,192 (B1a) 147,120 (B1b) 97,664 (B2a) 101,217 or (B2b) 76,128 S₀ − S₁ scans. In spectrum B2b, the orange and blue dashed traces are the best-fit deconvolved peaks whose integrated areas were used to determine fractions of β-sheet and helical populations in Table 3.

Fig. 7

The black and red traces are respectively the experimental and simulated REDOR S₁ ¹³CO spectra for protein in PC:PG membranes, with protein and experimental protein:lipid mol ratios of (A) FP34, 1:25; (B) N70, 1:30 or (C) FP-Hairpin, 1:30. Each S₁ spectrum represents the sum of contributions from natural abundance ¹³CO nuclei in the protein and the area of each simulated spectrum was scaled to that of the corresponding experimental spectrum. The orange, blue, and green traces in the bottom panels are respectively the calculated β-sheet/strand, helical, and coil backbone signals and the gray trace is the sidechain signal. The vertical lines are at the peak simulated ¹³CO chemical shifts of β-sheet/strand (right) or helical (left) signals.