Journal of Biochemistry Advance Access originally published online on January 3, 2009
Journal of Biochemistry 2009 145(3):299-307; doi:10.1093/jb/mvn180
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Turnip Mosaic Virus Genome-Linked Protein VPg Binds C-Terminal Region of Cap-Bound Initiation Factor 4E Orthologue Without Exhibiting Host Cellular Specificity
1Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094; 2Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 216-8511; 3Genomic Research Institute and Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan; and 4Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
*To whom correspondence should be addressed. Tel/Fax: +81-72-690-1069, E-mail: tomoo{at}gly.oups.ac.jp
Received October 10, 2008; Accepted November 26, 2008
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Abstract |
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To investigate the binding specificity of turnip mosaic virus (TuMV) viral protein-genome linked (VPg) with translation initiation factor 4E, we evaluated here the kinetic parameters for the interactions of human eIF4E, Caenorhabditis elegans IFE-3 and IFE-5 and Arabidopsis eIFiso4E, by surface plasmon resonance (SPR). The results indicated that TuMV VPg does not show a binding preference for Arabidopsis eIFiso4E, even though it is from a host species whereas the other eIF4E orthologues are not. Surprisingly, the effect of m7GTP on both the rate constants and equilibrium binding constants for the interactions of VPg differed for the four eIF4E orthologues. In the case of eIFiso4E and IFE-3, m7GTP increased kon, but for eIF4E and IFE-5, it decreased kon. To provide insight into the structural basis for these differences in VPg binding, tertiary structures of the eIF4E orthologues were predicted on the basis of the previously determined crystal structure of m7GpppA-bound human eIF4E. The results suggested that in cap-bound eIF4E orthologues, the VPg binds to the C-terminal region, which constitutes one side of the entrance to the cap-binding pocket, whereas in the cap-free state, VPg binds to the widely opened cap-binding pocket and its surrounding region. The binding of VPg to the C-terminal region was confirmed by the SPR analyses of N- or C-terminal residues-deleted eIF4E orthologues.
Key Words: 3D structure prediction, eIF4E, surface plasmon resonance, turnip mosaic virus, VPg
Abbreviations: 4EBP, eIF4E-binding protein; eIF, eukaryotic initiation factor; CM, carboxymethyled; eIF4E, eukaryotic initiation factor 4E; IRES, internal ribosome entry site; GST, glutathione-S-transferase; IPTG, isopropyl-β-D-thiogalactopyranoside; m7GTP, 7-methylguanosine 5'-triphosphate; m7GpppA, P1-7-methylguanosine-P3-adenosine- 5',5'-triphosphate; LB, Luria-Bertani; nCBP, novel cap-binding protein; PCR, polymerase chain reaction; P-20, poly(oxyethylene)sorbitan monolaulate (Tween 20); RU, resonance unit; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPR, surface plasmon resonance; TF, trigger factor; TuMV, turnip mosaic virus; VPg, viral protein linked to the genome