Department of Chemistry and Biochemistry, Hunter College of City University of New York, New York, New York 10065, United States.
Biochemistry. 2012 Feb 21;51(7):1388-95. doi: 10.1021/bi201929h. Epub 2012 Feb 9.
VPg of turnip mosaic virus (TuMV) was previously shown to interact with translation initiation factor eIFiso4F and play an important role in mRNA translation [Khan, M. A., et al. (2008) J. Biol. Chem.283, 1340-1349]. VPg competed with cap analogue for eIFiso4F binding and competitively inhibited cap-dependent translation and enhanced cap-independent translation to give viral RNA a significant competitive advantage. To gain further insight into the cap-independent process of initiation of protein synthesis, we examined the effect of PABP and/or eIF4B on the equilibrium and kinetics of binding of VPg to eIFiso4F. Equilibrium data showed the addition of PABP and/or eIF4B to eIFiso4F increased the binding affinity for VPg (K(d) = 24.3 ± 1.6 nM) as compared to that with eIFiso4F alone (K(d) = 81.3 ± 0.2.4 nM). Thermodynamic parameters showed that binding of VPg to eIFiso4F was enthalpy-driven and entropy-favorable with the addition of PABP and/or eIF4B. PABP and eIF4B decreased the entropic contribution by 67% for binding of VPg to eIFiso4F. The decrease in entropy involved in the formation of the eIFiso4F·4B·PABP-VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. The kinetic studies of eIFiso4F with VPg in the presence of PABP and eIF4B show 3-fold faster association (k(2) = 182 ± 9.0 s(-1)) compared to that with eIFiso4F alone (k(2) = 69.0 ± 1.5 s(-1)) . The dissociation rate was 3-fold slower (k(-2) = 6.5 ± 0.43 s(-1)) for eIFiso4F with VPg in the presence of PABP and eIF4B (k(-2) = 19.0 ± 0.9 s(-1)). The addition of PABP and eIF4B decreased the activation energy of eIFiso4F with VPg from 81.0 ± 3.0 to 44.0 ± 2.4 kJ/mol. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves to sequester initiation factors.
芜菁花叶病毒（TuMV）的 VPg 先前被证明与翻译起始因子 eIFiso4F 相互作用，并在 mRNA 翻译中发挥重要作用[Khan，M.A.等人。（2008）J. Biol. Chem.283，1340-1349]。VPg 与帽类似物竞争与 eIFiso4F 的结合，并竞争性地抑制帽依赖性翻译，增强帽非依赖性翻译，使病毒 RNA 具有显著的竞争优势。为了更深入地了解蛋白质合成起始的帽非依赖性过程，我们研究了 PABP 和/或 eIF4B 对 VPg 与 eIFiso4F 结合的平衡和动力学的影响。平衡数据表明，与单独的 eIFiso4F 相比，将 PABP 和/或 eIF4B 添加到 eIFiso4F 中增加了 VPg 的结合亲和力（K(d)=24.3±1.6 nM）。热力学参数表明，VPg 与 eIFiso4F 的结合是焓驱动的，并且在添加 PABP 和/或 eIF4B 时是熵有利的。PABP 和 eIF4B 使 VPg 与 eIFiso4F 结合的熵贡献降低了 67%。形成 eIFiso4F·4B·PABP-VPg 复合物所涉及的熵减少表明复合物形成和整体构象变化的疏水性相互作用减弱。在 PABP 和 eIF4B 的存在下，eIFiso4F 与 VPg 的动力学研究表明，与单独的 eIFiso4F 相比，结合速度提高了 3 倍（k(2)=182±9.0 s(-1))。在 PABP 和 eIF4B 的存在下，eIFiso4F 与 VPg 的解离速率降低了 3 倍（k(-2)=6.5±0.43 s(-1))（k(-2)=19.0±0.9 s(-1)）。添加 PABP 和 eIF4B 将 eIFiso4F 与 VPg 的活化能从 81.0±3.0 降低到 44.0±2.4 kJ/mol。这表明这两种蛋白质的存在导致快速、稳定的复合物，从而隔离起始因子。
