Abstract
Binding of phosphorylated eIFiso4E with viral genome-linked protein (VPg) of turnip mosaic virus was examined by stopped-flow, fluorescence, circular dichroism (CD) spectroscopy, and molecular docking analysis. Phosphorylation of eIFiso4E increased (4-fold) the binding rates as compared to unphosphorylated eIFiso4E with VPg. Stopped-flow kinetic studies of phosphorylated eIFiso4E with VPg showed a concentration-independent conformational change. The dissociation rate was about 3-fold slower for eIFiso4E∙VPg complex upon phosphorylation. Phosphorylation enhanced the association rates and lowered the dissociation rates for the eIFiso4E∙VPg binding, with having higher preferential binding to eIFiso4Ep. Binding rates for the interaction of eIFiso4Ep with VPg increased (6-fold) with an increase in temperature, 278 K to 298 K. The activation energies for binding of eIFiso4Ep and eIFiso4E with VPg were 37.2 ± 2.8 and 52.6 ± 3.6 kJ/mol, respectively. Phosphorylation decreased the activation energy for the binding of eIFiso4E to VPg. The reduced energy barrier suggests more stable platform for eIFiso4Ep∙VPg initiation complex formation, which was further supported by molecular docking analysis. Moreover, far-UV CD studies revealed that VPg formed complex with eIFiso4Ep with substantial change in the secondary structure. These results suggested that phosphorylation, not only reduced the energy barrier and dissociation rate but also enhanced binding rate, and an overall conformational change, which provides a more stable platform for efficient viral translation.
Highlights
Viruses depend on the translational apparatus of the host cells and have developed sophisticated mechanisms to suppress translation of cellular mRNAs whereas ensuring its own translation
To examine the effect of phosphorylation on VPg-dependent viral translation, we further investigated the binding rates of eIFiso4Ep interaction to VPg of turnip mosaic virus by stopped-flow experiments
Protein synthesis is a dynamic process in which protein-protein or protein-RNA binding and release occur for each cycle of translation initiation
Summary
Viruses depend on the translational apparatus of the host cells and have developed sophisticated mechanisms to suppress translation of cellular mRNAs whereas ensuring its own translation. The eIF4E or eIFiso4E complexes found in plants are key interacting partners of the VPg proteins found in many RNA viruses and are required for infection [7, 27]. Interaction of biological macromolecules VPg to eIF4E or its isoform eIFiso4E induces structural changes of the cap binding protein and diminishes the cap binding ability of initiation factors, resulting in the interruption of the formation of the translation initiation complex [15, 17]. Phosphorylation increased the translation by higher binding affinity of eIF4E with mRNA cap [35] These observations suggested that phosphorylation of eIF4E is Kinetic analysis of eIFiso4Ep VPg binding crucial for regulatory translational mechanism, which might mediate the functional interaction between viral VPg and host plant isoform eIFiso4E. To further examine the role of phosphorylation, we used stopped-flow and circular dichroism to dissect the kinetic mechanism and structural changes of eIFiso4Ep binding to VPg
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