NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII.
NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII.
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DOI：
10.1002/pro.2807
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发表时间：
2015-12
期刊：
影响因子：
--
通讯作者：
Skern T
中科院分区：
文献类型：
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作者：
Aumayr M;Fedosyuk S;Ruzicska K;Sousa-Blin C;Kontaxis G;Skern T
信使RNA通过包括真核起始因子（eIF）4 E（帽结合蛋白）、支架蛋白eIF 4G和RNA解旋酶eIF 4A的复合物募集到真核核糖体。为了关闭宿主细胞蛋白质合成，eIF 4G在小核糖核酸病毒感染期间被病毒编码的蛋白酶切割;该反应的结构基础及其由eIF 4 E的刺激尚不清楚。我们从结构和生物化学上研究了纯化的口蹄疫病毒（FMDV）前导蛋白酶（Lbpro）、人鼻病毒2型（HRV 2）2A蛋白酶（2Apro）和柯萨奇病毒B4（CVB 4）2Apro与纯化的eIF 4GII、eIF 4 E和eIF 4GII/eIF 4 E复合物的相互作用。使用核磁共振（NMR），我们完成了人eIF 4GII残基551-745的13 C/15 N连续骨架分配，并检查了它们与鼠eIF 4 E的结合。eIF 4GII 551 -745本质上是非结构化的，并且当与eIF 4 E结合时保持如此。用于确定化学计量和结合常数的NMR和生物物理技术显示，木瓜蛋白酶样Lbpro仅在eIF 4 E存在下与eIF 4GII 551 -745形成稳定的复合物，KD值在低纳摩尔范围内; Lbpro接触eIF 4GII和eIF 4 E。此外，来自HRV 2和CVB 4的不相关的胰凝乳蛋白酶样2Apro也与eIF 4GII/eIF 4 E构建稳定的复合物，但Kd值在低微摩尔范围内。HRV 2酶也与eIF 4 E形成稳定的复合物;然而，没有一种测试的蛋白酶与单独的eIF 4GII稳定复合。因此，这三种小核糖核酸病毒蛋白酶已经独立进化，以建立不同的三角形异源三聚体蛋白复合物，其可以主动靶向参与mRNA募集的核糖体，以确保有效的宿主细胞关闭。
Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap‐binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut‐off host–cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot‐and‐mouth disease virus (FMDV) leader proteinase (Lbpro), human rhinovirus 2 (HRV2) 2A proteinase (2Apro) and coxsackievirus B4 (CVB4) 2Apro with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed 13C/15N sequential backbone assignment of human eIF4GII residues 551–745 and examined their binding to murine eIF4E. eIF4GII551–745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain‐like Lbpro only forms a stable complex with eIF4GII551–745 in the presence of eIF4E, with K D values in the low nanomolar range; Lbpro contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin‐like 2Apro from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K D values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut‐off.