Ctures (NiV-G/ephrin-B3, NiV-G/ ephrin-B2) [32,50]. The superimposed structures of the G-H loop in the HeV-G/ ephrin-B2 complex and in unbound ephrin-B2 are presented on Figure 6B, revealing a total of three distinct conformations/ rotameric-forms of W122. During receptor binding, W122 1379592 is inserted into a groove on the HeV-G surface (Figure 6B). The unbound rotamer conformation doesn’t fit well because of the groove’s shape and localized negative charge. Rotating the sideCritical residues in the receptor-binding interface contribute to the distinct receptor-binding properties of NiV-G and HeV-GIt has been suggested that HeV-G has a lower than NiV-G affinity for its receptors and our data here provides a structural basis for this observation. [46] Indeed a comparison of the residues in the receptor-binding region of HeV-G with those of NiV-G reveals that three contact residues of NiV-G (V507, F458 and I401) are replaced in HeV-G with less hydrophobic ones (T507, Y458 and V401) (Figure 4). These residues participate in forming the receptor binding pockets for the hydrophobic Chebulagic acid web ephrin (G-H loop) residues P119, L121 and W122 respectively, and theHendra Virus Entry Mechanism Implied by StructureFigure 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. doi:10.1371/journal.pone.0048742.gFigure 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. doi:10.1371/journal.pone.0048742.gHendra Virus Entry Mechanism Implied by Structureup” and “latch-down” conformations depend on the strength of the hydrophobic Tunicamycin interactions within the core of the G-receptor interface. As discussed above, the HeV-G-receptor interface is less hydrophobic and, consequently, of somewhat lower affinity than its NiV-G-receptor counterpart. Accordingly, the dissociation rate is higher, with a 24195657 higher occupancy of the “latch-up” population.Structure-based mutagenesis reveals that the stability of the HeV-G ephrin complex is not stringently correlated with viral entry efficiencyTo explore the functional importance of the key interacting elements observed in the binding interface between HeV-G and the ephrin-B2 and -B3 receptors, a series of structure-based alanine substitutions were made in the full length, wild-type (WT) HeV-G, targeting various residues which make up the pockets that accommodate the ephrin-B2 G-H loop residues F117, P119, L121 and W122, or the ephrin-B3 G-H loop residues Y120, P122, L124 and W125. These HeV-G mutations included V401A, N402A, Q490A, W504A, E505A, G506A, Q559A, Y581A, and I588A. In addition, E501A and E533A mutations were also generated, which targeted the HeV-G-ephrin salt bridges at the periphery of the HeV-G-ephrin interface. We first examined whether the G-glycoprotein mutations effected the HeV-G/ephrin association as measured by coimmunoprecipitation. The series of G glycoprotein mutants were expressed in ephrin receptor negative cells either alone or together with their F glycoprotein partner. Cellular lysates were prepared and a series of precipitati.Ctures (NiV-G/ephrin-B3, NiV-G/ ephrin-B2) [32,50]. The superimposed structures of the G-H loop in the HeV-G/ ephrin-B2 complex and in unbound ephrin-B2 are presented on Figure 6B, revealing a total of three distinct conformations/ rotameric-forms of W122. During receptor binding, W122 1379592 is inserted into a groove on the HeV-G surface (Figure 6B). The unbound rotamer conformation doesn’t fit well because of the groove’s shape and localized negative charge. Rotating the sideCritical residues in the receptor-binding interface contribute to the distinct receptor-binding properties of NiV-G and HeV-GIt has been suggested that HeV-G has a lower than NiV-G affinity for its receptors and our data here provides a structural basis for this observation. [46] Indeed a comparison of the residues in the receptor-binding region of HeV-G with those of NiV-G reveals that three contact residues of NiV-G (V507, F458 and I401) are replaced in HeV-G with less hydrophobic ones (T507, Y458 and V401) (Figure 4). These residues participate in forming the receptor binding pockets for the hydrophobic ephrin (G-H loop) residues P119, L121 and W122 respectively, and theHendra Virus Entry Mechanism Implied by StructureFigure 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. doi:10.1371/journal.pone.0048742.gFigure 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. doi:10.1371/journal.pone.0048742.gHendra Virus Entry Mechanism Implied by Structureup” and “latch-down” conformations depend on the strength of the hydrophobic interactions within the core of the G-receptor interface. As discussed above, the HeV-G-receptor interface is less hydrophobic and, consequently, of somewhat lower affinity than its NiV-G-receptor counterpart. Accordingly, the dissociation rate is higher, with a 24195657 higher occupancy of the “latch-up” population.Structure-based mutagenesis reveals that the stability of the HeV-G ephrin complex is not stringently correlated with viral entry efficiencyTo explore the functional importance of the key interacting elements observed in the binding interface between HeV-G and the ephrin-B2 and -B3 receptors, a series of structure-based alanine substitutions were made in the full length, wild-type (WT) HeV-G, targeting various residues which make up the pockets that accommodate the ephrin-B2 G-H loop residues F117, P119, L121 and W122, or the ephrin-B3 G-H loop residues Y120, P122, L124 and W125. These HeV-G mutations included V401A, N402A, Q490A, W504A, E505A, G506A, Q559A, Y581A, and I588A. In addition, E501A and E533A mutations were also generated, which targeted the HeV-G-ephrin salt bridges at the periphery of the HeV-G-ephrin interface. We first examined whether the G-glycoprotein mutations effected the HeV-G/ephrin association as measured by coimmunoprecipitation. The series of G glycoprotein mutants were expressed in ephrin receptor negative cells either alone or together with their F glycoprotein partner. Cellular lysates were prepared and a series of precipitati.
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