Figure 4
Binding to the membrane-inserted epitope.
(a) Model of the recognition of the epitope by 10E8 and 4E10 Fab in membranes. Left: Relative orientation of 10E8 (gray) with respect to 4E10 (PDB entry code 2FX7, cyan) in membranes. The position of the peptide is such that occupies an oblique orientation as in Irimia et al.16. The position of 10E8 was calculated after superimposing residues 673-678 of the peptide bound in the structures with 10E8 or 4E10 (RMSD = 0.25 Å). The angle between the two Fab chains was calculated with CHIMERA. The lipid molecule bound to 4E10 was obtained from the structure (PDB entry 4XCN). Carbon, oxygen and phosphate atoms of the lipid are depicted with blue, red and yellow sticks, respectively. Right: Several phosphate ions (red and yellow) are bound to 10E8, suggesting a putative orientation of the antibody with respect to the surface of the viral membrane (represented with a dotted line). 10E8 (gray) and 4E10 (cyan) Fabs are shown in the same orientation as in the left panel. Residue Trp100bHC of 10E8 and the molecule of lipid bound to 4E10 are depicted in magenta and blue, respectively. Inset: Close-up view of the circled region. (b) Photo-cross-linking of Fab 10E8 with peptides H(686) and H(693) inserted in membranes. Protein bands were detected by western-blot. Other experimental conditions are identical to those described in Fig. 3c. (c) Membrane insertion of the 10E8 CDRH3 apex probed by NBD fluorescence. Top panels: Spectra of NBD emission were measured upon incubation of NBD-Fab in solution (red traces), with liposomes devoid of peptide (black solid traces) or with LUV-peptide complexes that contained increasing amounts of H(686) or H(693) (black dotted traces in left and right panels, respectively). Bottom panels: Fractional changes (top) and maximum shifts (bottom) of NBD emission deduced from the spectra. The initial fluorescence (F0) was determined from NBD-Fab in buffer samples (red traces). Final fluorescence values (F) were measured after the addition of LUV-peptide complexes (black traces). Lipid concentration was fixed at 250 μM. Means ± S.D. of three independent experiments are represented. The lines correspond to the best fit of the experimental values to a hyperbolic function.
