Ebolavirus Entry Requires a Compact Hydrophobic Fist at the Tip of the Fusion Loop

Observed changes in chemical environment induced by alanine mutations. Assigned HSQC spectra of WT (a) and L529A I544A mutant (b) Ebov FLs in DPC micelles at pH 5.5. (c) ¹H and ¹⁵N chemical shift differences between L529A I544A and WT Ebov FLs. Ala mutation sites are marked with red circles. Proline residues and other residues that could not be assigned are marked with x. Chemical shift differences were combined according to Δδ_comp = [Δδ_HN² + (Δδ_N/6.25)²]^1/2 (65).

Observed changes in chemical environment induced by alanine mutations. Shown are assigned HSQC spectra of the L529A (a) and I544A (b) mutant Ebov FLs in DPC micelles at pH 5.5. Also shown are the ¹H and ¹⁵N chemical shift differences between L529A (c) or I544A (d) mutant and WT Ebov FLs. Proline residues and other residues that could not be assigned are marked with x. Mutation sites are labeled with a red circle. All measurements were carried out in DPC micelles at pH 5.5.

NMR structures of the WT (left) and I544A (center) and L529A I544A (right) mutant Ebov FLs in DPC micelles at pH 5.5. The 20 lowest-energy conformers are rendered for the WT (a), I544A (b), and L529A I544A (c) Ebov FLs. Irregularly structured loops and turn regions are shown in gray, the α-helix in green, the β-sheet in blue, and the disulfide-linked Cys511 and Cys556 in orange. The lowest-energy conformers are shown in a forward-facing view of the fusion loop tip (residues I527 to L547) for the WT (d), I544A mutant (e), and L529A I544A mutant (f). Residues 529, 535, and 544 are shown in a stick representation and colored blue, bright orange, and purple, respectively. Panels g to i show a hydrophobic surface representation at 20% transparency to reveal a hydrophobic scaffold in stick representation of the WT (g) and I544A (h) and L529A I544A (i) mutants. Residues mutated to Ala are shown in yellow. Heteronuclear NOEs and R1 and R2 relaxation rates are presented for the WT at pH 7.0 and 5.5 and the I544A and L529A I544A mutants in the relaxation data in the supplemental material.

Binding of WT and mutant FLs to lipid bilayers measured by ITC. (a) Ebov FL WT at pH 5.0; (b) Ebov FL WT at pH 7.4; (c) L529A mutant FL at pH 5.0; (d) I544A mutant FL at pH 5.0; (e) L529A I544A double mutant FL at pH 5.0. One exemplary titration is shown for each condition. At least two titrations were performed for each condition.

Depth of membrane insertion by tryptophan fluorescence quenching. (a and b) Fluorescence quenching profiles of Ebov FLs containing a single Trp at positions 518 (green), 531 (blue), 534 (cyan), and 543 (red) for the WT (a) and L529A mutant (b). Profiles were generated by plotting relative fluorescence F(d_Q)/F₀ as a function of the Br-quencher distance from the bilayer center. Experimental data were fit using the distribution analysis method (lines). A 5 μM concentration of peptide was incubated with 500 μM SUVs composed of POPC-POPG at 85:15 (control) or 55:15:30, where 30 mol% lipid was 6,7-, 9,10-, or 11,12-bromo PC. (c) Sequences of single tryptophan mutants used in the Trp fluorescence quenching experiments. Alanine mutations are highlighted in red, single Trp mutations in green, and phenylalanines in yellow. (d) Lipid mixing of single Trp and the Phe double mutations in the WT backbone confirms that FL constructs with Trp residues at positions 518, 531, 534, and 543 behave like the WT and therefore could be used for fluorescence quenching analyses. F535W was not used for fluorescence quenching but is included here to demonstrate the importance of this residue, which contributes to the triad of residues forming the structural scaffold of the FL fist structure. FLs (5 μM) were tested for fusion at pH 5.0 with POPC-POPG (85:15) liposomes (100 μM) and normalized to the activity of the WT. All results are from experiments repeated in triplicate with at least two preparations of liposomes. Error bars indicate standard deviations.

Molecular dynamics simulations showing insertion of Ebov FLs in POPC-POPG bilayers. (a to c) Atomistic simulations of the Ebov FL WT at pH 5.0 (a) and the Ebov FL I544A (b) and L529A I544A (c) mutants at pH 5.0, all rendered after 400 ns of simulation. The I544A mutant displayed a “flatter” structure but was still membrane associated, while the L529A I544A mutant was both less structured and significantly less inserted (P < 10⁻⁵). Coarse-grained simulations show the binding of the WT FL to a lipid bilayer starting from a solution conformation of WT Ebov FL at time 0 (d) to form a membrane-bound structure at 3 μs. The hydrophobic region, A525 to I544, is colored green, while the other residues are shown in red for the WT, magenta for the I544A mutant, and blue for the L529A I544A mutant. Phospholipid head groups are in orange, and in atomistic simulations, the first leaflet of the lipid bilayer is shown in a line representation. Movies of atomistic simulations of the WT (Video S1), the I544A mutant (Video S2), and the L529A I544A mutant (Video S3) insertions are shown in the supplemental material.