Structural basis for mRNA cap-binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods
Structural basis for mRNA cap-binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods
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DOI：
10.1016/j.bbapap.2005.07.023
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发表时间：
2005-12-01
影响因子：
3.2
通讯作者：
Ishida, T
中科院分区：
文献类型：
--
作者：
Tomoo, K;Matsushita, Y;Ishida, T
利用Trp 73残基位于eIF 4 E的4 E-BP结合位点附近的优势，采用Tip荧光滴定法研究了4 E-BP异构体与eIF 4 E的相互作用。虽然在三种4 E-BP亚型的结合常数之间没有观察到显著差异，但显示出4 E-BP 2比4 E-BP 1和4 E-BP 3更优先结合，这可能是由于4 EBP 2特异性LDRR（60-63）序列对与eIF 4 E结合的影响。相比之下，表面等离子体共振（SPR）分析显示4 E-BP 1的结合偏好，尽管同种型之间的差异也不显著。这种与荧光分析的不一致可能是由于相互作用的不同观察点，即，局部和整体的相互作用观察的荧光和SPR方法，分别。为了阐明这些光谱结果的结构基础，通过X射线衍射方法分析了三元复合物的晶体结构：in 7 GpppA-eIF 4 E-4 E-BP 1片段（Thr 36-Thr 70）。在2.1埃分辨率下的晶体结构分析显示，4 E-BP 1片段，归属于Pro 47-Pro 66肽部分，采用涉及p折叠和a-螺旋结构的反向L形构象，并且通过亲水和疏水相互作用位于钟形eIF 4 E的柄的根部。基于观察到的结合模式，与三种4 E-BP亚型可能的相互作用进行了讨论。另一方面，由于与先前确定的m（7）GPPpA-eIF 4 E-4 E二元复合物的晶体结构比较显示4 E-BP I片段的对接不显著影响eIF 4 E的总体三级结构和帽结合支架，因此通过分子动力学（MD）研究4 E-BP I对eIF 4 E的帽结合的动态调节。模拟因此，模拟表明（i）4 E-BP 1肽的螺旋区对于与eIF 4 E的结合是重要的，（ii）帽结构的存在稳定了eIF 4 E与4 E-BP的结合，（iii）4 E-BP的结合稳定了eIF 4 E的帽结合口袋，和（iv）单独的Ser 67的磷酸化不诱导4 E-BP与eIF 4 E的分离，但增加了4 E-BP的结构刚性。这些结果为4 E-BP调控eIF 4 E的mRNA帽结合提供了结构基础。(c)2005 Elsevier B.V保留所有权利。
Taking advantage of the Trp73 residue located close to the 4E-BP binding site of eIF4E, the interaction between the 4E-BP isoform and eIF4E was investigated by the Tip fluorescence titration method. Although no significant difference was observed among the association constants of three 4E-BP isoforms, the binding preference of 4E-BP2 over 4E-BP1 and -BP3 was shown, probably due to the effect of a 4EBP2-specific LDRR (60-63) sequence for the binding with eIF4E. By contrast, surface plasmon resonance (SPR) analyses showed the binding preference of 4E-BP1, although the difference among the isoforms was also not significant. This inconsistency with fluorescence analysis likely resulted from the different observation points of the interaction, i.e., local and overall interactions observed by the fluorescence and SPR methods, respectively. To clarify the structural basis for these spectroscopic results, the crystal structure of the ternary complex:of in 7 GpppA-eIF4E-4E-BP1 fragment (Thr36-Thr70) was analyzed by the X-ray diffraction method. Crystal structure analysis at 2.1 angstrom resolution revealed that the 4E-BP1 fragment, assigned to the Pro47-Pro66 peptide moiety, adopted a reverse L-shaped conformation involving the p sheet and a-helical structures and was located at the root of the handle of the temple-bell-shaped eIF4E through hydrophilic and hydrophobic interactions. Based on the observed binding mode, possible interactions with the three 4E-BP isoforms have been discussed. On the other hand, since the crystal structural comparison with the previously determined m(7)GPPpA-eIF4E-4E binary complex showed that the docking of the 4E-BP I fragment does not significantly affect the overall tertiary structure and cap-binding scaffold of eIF4E, the dynamic regulation of the cap-binding of eIF4E by 4E-BP 1 was investigated by molecular dynamics (MD). simulations. Consequently, the simulation suggested that (i) the helical region of the 4E-BP1 peptide is important for the binding with eIF4E, (ii) the existence of a cap structure stabilizes the binding of eIF4E with 4E-BP, (iii) the binding of 4E-BP stabilizes the cap binding pocket of eIF4E, and (iv) the phosphorylation of Ser67 alone does not induce the separation of 4E-BP from eIF4E, but increases the structural rigidity of 4E-BP. These results provide the structural basis for the mRNA cap-binding regulation of eIF4E by 4E-BP. (c) 2005 Elsevier B.V All rights reserved.