Suggests how receptor binding by G could be a trigger for F-activation: Virus in the prefusion state possesses F is in its “metastable” (high-energy) state presumably in association with the intact tetrameric HeV-G [24]. Receptor binding causes subtle conformational changes at the HeV-G/ephrin interface which are relayed to the side of the G beta propeller to alter the weak dimeric HeV-G interface and may cause dissociation of the HeV-G tetramers. Following this, HeV-F is triggered and allowed to proceed through a structural transformation into the lower-energy “activated” state which facilitates the membrane fusion process, either by release of inhibition (referred to as the clamp model) or by promotion with the dissociation of HeV-G tetramers (referred to as the provocateur model) [47,48]. However, we recently extensively characterize several forms of trimeric soluble henipavirus F glycoprotein [49] and reported that it appears that the henipavirus F glycoprotein is expressed in an apparent pre-fusion conformation in the absence of the coexpression of its partner G glycoprotein, strongly suggestion that the `clamp model’ as defined may not be accurate. Here, to explore these models further, we analyzed the structures of the four distinct HeV-G/ephrin-B2 complexes within the crystal asymmetric unit and carried out a series of structure-based mutagenesis experiments in HeV-G.Figure 2. Structure of the HeV-G/ephrin-B2 complex. The HeV-G and ephrin-B2 molecule are colored in yellow and purple, MedChemExpress (-)-Indolactam V respectively. Ephrin-B2 sits on top face of the HeV-G b-propeller. The G-H loop of ephrin-B2 extends into the central cavity of HeV-G’s b-propeller barrel. doi:10.1371/journal.pone.0048742.gEphrin-B2 (S27-D167) used in our structural studies here was expressed in stably-transfected human HEK293 cells. Thus, its glycosylation pattern is more physiologically relevant than in the previously published structures, which used ephrin expressed in either yeast, bacteria or in the presence of glycosylation inhibitor. In addition to the previously observed glycosylation site N36 [32,44], we now observe another glycosylation site – N139, which is consistent with sequence-based predictions. Ephrin-B2 undergoes very minor conformational changes upon binding to 24195657 HeV-G and its structure in the HeV-G/ephrin-B2 complex can be superimposed on the unbound ephrin-B2 structure with the root mean square deviation (RMSD) between ?equivalent Ca positions of approximately 0.5 A [44,45]. The only region on ephrin-B2 that shows some rearrangements upon HeVG binding is the G-H loop. These conformational changes within this ephrin region are necessary to fit its four hydrophobic residues (F, P, L and W) within the G-H loop into their binding pockets on the HeV-G surface.W122 serves as a “latch” during virus-receptor associationVirus-receptor association and dissociation is a dynamic process, which is usually difficult to study using conventional crystallographic methods; however, our crystals provide some very useful snapshots of this process. Specifically, we observed two rotameric forms of W122 in ephrin-B2 among the four HeV-G/ ephrin-B2 complexes in the asymmetric unit (Figure 6A). In two of the complexes the indole group lies parallel to the HeV-G binding face (“down”). In the other two complexes this group stands MedChemExpress LY2409021 perpendicular to the HeV-G binding face (“up”). Interestingly, the “up” rotamer of this tryptophan residue is not observed in any of the related stru.Suggests how receptor binding by G could be a trigger for F-activation: Virus in the prefusion state possesses F is in its “metastable” (high-energy) state presumably in association with the intact tetrameric HeV-G [24]. Receptor binding causes subtle conformational changes at the HeV-G/ephrin interface which are relayed to the side of the G beta propeller to alter the weak dimeric HeV-G interface and may cause dissociation of the HeV-G tetramers. Following this, HeV-F is triggered and allowed to proceed through a structural transformation into the lower-energy “activated” state which facilitates the membrane fusion process, either by release of inhibition (referred to as the clamp model) or by promotion with the dissociation of HeV-G tetramers (referred to as the provocateur model) [47,48]. However, we recently extensively characterize several forms of trimeric soluble henipavirus F glycoprotein [49] and reported that it appears that the henipavirus F glycoprotein is expressed in an apparent pre-fusion conformation in the absence of the coexpression of its partner G glycoprotein, strongly suggestion that the `clamp model’ as defined may not be accurate. Here, to explore these models further, we analyzed the structures of the four distinct HeV-G/ephrin-B2 complexes within the crystal asymmetric unit and carried out a series of structure-based mutagenesis experiments in HeV-G.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 b-propeller. The G-H loop of ephrin-B2 extends into the central cavity of HeV-G’s b-propeller barrel. doi:10.1371/journal.pone.0048742.gEphrin-B2 (S27-D167) used in our structural studies here was expressed in stably-transfected human HEK293 cells. Thus, its glycosylation pattern is more physiologically relevant than in the previously published structures, which used ephrin expressed in either yeast, bacteria or in the presence of glycosylation inhibitor. In addition to the previously observed glycosylation site N36 [32,44], we now observe another glycosylation site – N139, which is consistent with sequence-based predictions. Ephrin-B2 undergoes very minor conformational changes upon binding to 24195657 HeV-G and its structure in the HeV-G/ephrin-B2 complex can be superimposed on the unbound ephrin-B2 structure with the root mean square deviation (RMSD) between ?equivalent Ca positions of approximately 0.5 A [44,45]. The only region on ephrin-B2 that shows some rearrangements upon HeVG binding is the G-H loop. These conformational changes within this ephrin region are necessary to fit its four hydrophobic residues (F, P, L and W) within the G-H loop into their binding pockets on the HeV-G surface.W122 serves as a “latch” during virus-receptor associationVirus-receptor association and dissociation is a dynamic process, which is usually difficult to study using conventional crystallographic methods; however, our crystals provide some very useful snapshots of this process. Specifically, we observed two rotameric forms of W122 in ephrin-B2 among the four HeV-G/ ephrin-B2 complexes in the asymmetric unit (Figure 6A). In two of the complexes the indole group lies parallel to the HeV-G binding face (“down”). In the other two complexes this group stands perpendicular to the HeV-G binding face (“up”). Interestingly, the “up” rotamer of this tryptophan residue is not observed in any of the related stru.
