Two Key Residues in EphrinB3 Are Critical for Its Use as an Alternative Receptor for Nipah Virus

Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, California, USA.
PLoS Pathogens (Impact Factor: 8.06). 03/2006; 2(2):e7. DOI: 10.1371/journal.ppat.0020007
Source: PubMed

ABSTRACT EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.

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    ABSTRACT: The dynamical properties of water at protein-water interfaces are unlike those in the bulk. Here we utilize molecular dynamics simulations to study water dynamics in interstitial regions between two proteins. We consider two natural protein-protein complexes, one in which the Nipah virus G protein binds to cellular ephrin B2, and the other in which the same G protein binds to ephrin B3. While the two complexes are structurally similar, the two ephrins share only a modest sequence identity of $\sim$50\%. X-ray crystallography also suggests that these interfaces are fairly extensive, and contain exceptionally large amounts of waters. We find that while the interstitial waters tend to occupy crystallographic sites, most waters exhibit residence times of less than hundred picoseconds in the interstitial region. We also find that while the differences in the sequence of the two ephrins result in quantitative differences in the dynamics of interstitial waters, the trends in the shifts with respect to bulk values are similar. Despite the high wetness of the protein-protein interfaces, the dynamics of interstitial waters are considerably slower compared to the bulk - the interstitial waters diffuse an order of magnitude slower, have 2-3 fold longer hydrogen bond lifetimes and 2-1000 fold slower dipole relaxation rates. To understand the role of interstitial waters, we examine how implicit solvent models compare against explicit solvent models in producing ephrin-induced shifts in the G conformational density. Ephrin-induced shifts in the G conformational density are critical to the allosteric activation of another viral protein that mediates fusion. We find that in comparison with the explicit solvent model, the implicit solvent model predicts a more compact G--B2 interface, presumably because of the absence of discrete waters at the G--B2 interface. Simultaneously, we find that the two models yield strikingly different induced changes in the G conformational density, even for those residues whose conformational densities in the apo state are unaffected by the treatment of the bulk solvent. Together, these results show that the explicit treatment of interstitial water molecules is necessary for a proper description of allosteric transitions.
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