Gc interactions with lipids and post-fusion structure.

<p>A) scFv A5 specifically inhibits liposome binding by rGc as measured by SPR. The liposomes were immobilized on an SPR chip (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005813#sec013" target="_blank">Methods</a>) and Gc alone or together with scFv A5 at increased concentrations was injected to measure binding (top panel). The same experiment with an unrelated, non-binding scFv is provided as control in the lower panel. B) Left panel: Electron micrographs of negative stained samples of rGc show long thin rods with dimensions comparable to the neutral pH structure shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005813#ppat.1005813.g001" target="_blank">Fig 1B and 1C</a>. Right Panel: Incubation of rGc with liposomes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005813#sec013" target="_blank">Materials and Methods</a> for lipid composition) at low pH results in rGc insertion into the membranes, with a concomitant change in aspect to resemble class II viral fusion proteins inserted into liposomes in their post-fusion, trimeric conformation (as shown previously for alphaviruses and flaviviruses). C) The structure of the Gc trimer crystallized at pH 6.5 (W115H mutant) displays the typical class II post-fusion conformation. The protomer in the foreground is shown in ribbons colored by domains, with the single glycosylation site at Asn280 in sticks, as well as key residues at the tip of domain II. The two protomers in the background are shown as surfaces, one colored in grey and the other by domains, with the glycan surface in green. The structure determination used a W115H mutant, but the representation shows the location of the mutated tryptophan (labeled). The trimer axis is drawn in black. D) Conformational transition of rGc. The neutral pH monomeric form colored gray and the low pH trimer subunit colored according to domains were superposed on domain I and are shown in two views to highlight the overall reorganization of the molecule. The left panel shows the axis (nearly normal to the plane of the Figure) about which domain III pivots by more than 120 degrees. The right panel shows the 26 degrees hinge of domain II about the domain I/II junction. E) Close up view (from the top) of the tip of domain II showing the structuring role of Asn118. The polypeptide chain is shown in yellow except for the <i>cd</i> loop (orange), with non-carbon atoms in different color (nitrogen, oxygen and sulfur in blue, red and green, respectively). Asn118 is highlighted in white sticks with an orange outline. Its side chain carboxamide group donates two hydrogen bonds (yellow dots) to the main chain carbonyls of Cys122 and Gly124, and accepts a hydrogen bond from the main chain amide of Ser114. In addition, the main chain carbonyl of Asn118 accepts two hydrogen bonds from the <i>ij</i> loop, from the main chain amide groups of Ala251 and Thr252. Note that Asn118 is framed by disulfides 4 (immediately upstream) and 1 (downstream), which connect the <i>cd</i> loop to the <i>ij</i> and <i>bc</i> loops, respectively. Trp115 and Phe250, discussed in the paper, are displayed in sticks, as a guide. F) Cartoon to show the swapping of β-strand A₀ during trimerization. The left panel shows domain I in the pre-fusion conformation, with inner and outer β-sheets in yellow and green, respectively. The short A₀ strand runs antiparallel to C₀ at one edge of the outer β-sheet (indicated in yellow to highlight that it becomes part of the inner sheet in the post-fusion form). In the trimer, the A₀ strand swaps to the adjacent domain I (depicted with inner and outer β sheets in pink and red, respectively), to run antiparallel to the B₀ strand in the inner β-sheet. For clarity, the front subunit in the trimer (which would have inner and outer sheets in pale and dark blue) was removed, leaving only its A₀ strand, which inserts into the β-sandwich of the yellow/green domain (on which the monomer was superposed for this view). The “pink” A₀ strand inserts into the blue subunit (not displayed for clarity). Downstream strand I₀, the domain I-III linker runs alongside the inserted A₀ strand in the trimer, swapping also domain III, which is further downstream (see panel C). The limits of strand A₀ are labeled, to show what residues rearrange (see also the secondary structure diagram in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005813#ppat.1005813.g001" target="_blank">Fig 1D</a>).