Functional architecture of HCVIRES domain II stabilized by divalent metal ions in the crystal and in solution
Functional architecture of HCVIRES domain II stabilized by divalent metal ions in the crystal and in solution
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DOI：
10.1002/anie.200603807
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Hermann, Thomas
中科院分区：
文献类型：
--
作者：
Dibrov, Sergey M.;Johnston-Cox, Hillary;Hermann, Thomas
丙型肝炎病毒（HCV）的RNA基因组包含一个内部核糖体进入位点（IRES），它与宿主细胞40S核糖体亚基结合，并在缺乏大多数起始因子的情况下启动蛋白质翻译。[1,2]核糖体亚基向HCV RNA的募集是由IRES-40S相互作用的高亲和力驱动的IRES序列采用高度有序的二级结构（图1a）IRES的三维结构由独立折叠的RNA结构域主导通过低温电子显微镜（cryo-EM）研究了IRES-40S复合物的结构，揭示了RNA的整体形状。[6,7]通过晶体学和核磁共振光谱已经确定了单个子域的高分辨率结构，包括II[8]和IIIa-e[9-12]。Cryo-EM研究显示，在HCV翻译中起重要结构作用的结构域ii[13]采用l形构象，引导顶端发夹环iib与P位点附近的核糖体E位点重叠domainII的结合诱导40S头部[7]的构象改变，关闭信使RNA （mRNA）的结合裂缝[6]核磁共振研究表明，亚结构域iia可能是一种柔性铰链，其弯曲状态通过二价金属离子的结合而稳定为了研究IRES结构域II结的分子结构及其金属离子依赖的稳定性，我们使用了x射线晶体学和结构引导下的荧光标记结合。用于x射线晶体结构测定（见辅助信息中的图S4和表S1），使用含有HCV IRES残基49-69和100-115的寡核苷酸（IIa-1）（图1）。RNA的整体结构揭示了IIa子结构域的弯曲结构。位于内部凸起侧面的两个阀杆以直角布置（图1b）
The RNA genome of the hepatitis C virus (HCV) contains an internal ribosome entry site (IRES), which binds to the hostcell 40S ribosomal subunit and initiates protein translation in the absence of most initiation factors.[1, 2] Recruitment of the ribosomal subunit to the HCV RNA is driven by the high affinity of the IRES–40S interaction.[3] The IRES sequence adopts a highly ordered secondary structure (Figure 1a).[4] The three-dimensional architecture of the IRES is dominated by independently folding RNA domains.[5] Structures of IRES–40S complexes have been studied by cryo-electron microscopy (cryo-EM) revealing the overall shape of the RNA.[6, 7] Higher-resolution structures of individual subdomains, including II [8] and IIIa–e,[9–12] have been determined by crystallography and NMR spectroscopy. Cryo-EM studies revealed that the domainII, which plays an important structural role in HCV translation,[13] adopts an L-shaped conformation that directs the apical hairpin loopIIb to overlap with the ribosomal E site in the proximity of the P site.[6] Binding of domainII induces a conformational change in the 40S head [7] and closes the messenger RNA (mRNA) binding cleft.[6] NMR studies suggested that subdomainIIa might be a flexible hinge whose bent state is stabilized by binding of divalent metal ions.[8] To investigate the molecular architecture of the IRES domain II kink and its metal-ion-dependent stabilization, we have used X-ray crystallography and structure-guided incorporation of fluorescent labels. For X-ray crystal structure determination (see Figure S4 and Table S1 in the Supporting Information), an oligonucleotide (IIa-1) was used that contained residues 49–69 and 100–115 of the HCV IRES (Figure1). The overall structure of the RNA revealed a bent architecture for the IIa subdomain. The two stems that are flanking the internal bulge are arranged at a right angle (Figure 1 b).[14]