Figure 1.
The secondary structure elements of the two molecules are colored in cyan and green. The axes of the two six-blade β-propellers are approximately perpendicular to each other. Disulfide bonds are illustrated as yellow sticks. Asparagine-linked carbohydrate modifications (glycosylations) are illustrated as grey spheres.
Figure 2.
Structure of the HeV-G/ephrin-B2 complex.
The HeV-G and ephrin-B2 molecule are colored in yellow and purple, respectively. Ephrin-B2 sits on top face of the HeV-G β-propeller. The G-H loop of ephrin-B2 extends into the central cavity of HeV-G's β-propeller barrel.
Figure 3.
Four hydrophobic residues of the ephrin-B2 G-H loop insert in four hydrophobic HeV-G pockets.
The four ephrin residues (F117, P119, L121 and W122) are illustrated as purple sticks. The HeV-G pockets are shown as a yellow surface. The residues defining these pockets are shown as yellow lines and are labeled.
Figure 4.
Comparison of the NiV-G/ephrin-B3 and HeV-G/ephrin-B2 interfaces.
HeV-G residues are colored in yellow; NiV-G is in cyan. The changes V/T507, F/Y458, I/V401 alter the hydrophobicity of the interface and thus contribute to the different receptor-binding affinities of NiV-G and HeV-G.
Figure 5.
Rearrangement of the receptor binding face of HeV-G upon ephrin-B2 binding.
Complexed HeV-G (yellow) is superimposed with unbound HeV-Gs (green and cyan, which represent two different molecules within the same asymmetric unit). The interface between the two HeV-G molecules within the unbound HeV-G homodimer is colored in red. The G-H loop of ephrin-B2 is colored in purple. The ephrin-B2-contacting regions of HeV-G are colored in pink. Receptor binding causes a dramatic conformational change in the B6S2-B6S3 loop region, which then disrupts the HeV-G homodimerization interface observed in the unbound HeV-G.
Figure 6.
Two rotameric W122 (ephrin-B2) conformations in the HeV-G/ephrin-B2 complex, both of which are distinct from the W122 conformation in unbound ephrin-B2. Ephrin-B2 is shown in silver in the unbound state, and in yellow or cyan in the two complexes with HeV-G (A). W122 transforms from the initial unbound conformation to an intermediate conformation upon binding to HeV-G due to steric and electrostatic constrains, then adopts its final conformation via stabilizing van der Waals interactions with HeV-G. W122 is shown as yellow sticks. G is shown as a surface colored according to its electrostatic potential (B). “Latch up” and “latch down” conformations of ephrin-B2-W122 mediate the association and dissociation of the HeV-G/ephrin-B2 complex. Ephrin-B2-W122 is shown in yellow, HeV-G - in grey (C).
Figure 7.
Expression and receptor binding activity of structure-based HeV-G mutations.
The various alanine substitution mutants or wild-type (WT) HeV-G were transiently expressed in the absence (A) or presence (B) of HeV-F in HeLa-USU cells. Cell lysates were prepared and equal amounts were co-precipitated with recombinant ephrin-B2/Fc or ephrin-B3/Fc, or directly immunoprecipitated with polyclonal G-specific antibodies (control), followed by protein G Sepharose beads. The precipitated samples were processed and analyzed by 4 to 20% gradient SDS-PAGE and Western blotting with HeV- G-specific antiserum. This experiment was repeated three times and one representative experiment is shown.
Figure 8.
Effect of structure-based HeV-G mutations on viral entry.
Luciferase-encoding HIV-1 based pseudovirus stocks were prepared in 293T cells using wild-type (WT) or alanine substitution mutants of HeV-G with the HeV-F by expression plasmid transfection together with pNL4-3-Luc-E-R+ as described in Methods. Each pseudovirus stock preparation was analyzed by p24 quantification and equal amounts of virus particles were used to infect target cells, either Hela-USU cells expressing ephrin-B2 (A) or ephrin-B3 (B), and performed in triplicate. Cells were incubated for 48 hr following infection and processed for luciferase activity quantification using a Centro LB 960 Microplate Luminometer (Berthold Technologies). This experiment was repeated six times and a representative experiment is shown. (C) Incorporation of the HeV F and wild-type and mutant G glycoproteins into pseudovirus particles was assessed by Western blot of lysates prepared from p24 normalized amounts of the same purified virus particles used in Panels A and B. HeV G was detected with a cross-reactive polyclonal mouse antiserum to HeV G and HeV F was detected with a rabbit polyclonal F1 specific antiserum as described in the Methods.
Figure 9.
Interaction of HeV-G mutants with HeV-F.
(A) HeV-G mutants were co expressed with S tagged HeV-F in HeLa-USU cells. Lysates were immunoprecipitated (IP) with rabbit polyclonal G-specific antiserum to evaluate total G expression (top panel) or S agarose to evaluate total F expression (bottom panel) and co-precipitation of G (middle panel). The precipitated products were analyzed on SDS-PAGE under reducing conditions and then western blot analyzed with F (bottom panel) or G (top and middle panel) specific mouse mAbs. The western blot result of one of three independent experiments is shown in (B). The relative HeV-F binding ability of each HeV-G mutant is shown in comparison to that of WT HeV-G and normalized to total HeV-G expression and the means of three independent experiments are shown. Error bars represent the ranges. The results were calculated using values obtained from digital densitometric measurements of the images.