The Structure of the Human Respiratory Syncytial Virus M2-1 Protein Bound to the Interaction Domain of the Phosphoprotein P Defines the Orientation of the Complex.
The Structure of the Human Respiratory Syncytial Virus M2-1 Protein Bound to the Interaction Domain of the Phosphoprotein P Defines the Orientation of the Complex.
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DOI：
10.1128/mbio.01554-18
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发表时间：
2018-11-13
期刊：
影响因子：
6.4
通讯作者：
Edwards TA
中科院分区：
文献类型：
--
作者：
Selvaraj M;Yegambaram K;Todd EJAA;Richard CA;Dods RL;Pangratiou GM;Trinh CH;Moul SL;Murphy JC;Mankouri J;Éléouët JF;Barr JN;Edwards TA
人呼吸道合胞病毒（HRSV）是呼吸道疾病的主要原因，特别是在年轻人，老年人和免疫功能低下的人中，并且还与哮喘的发展有关。HRSV复制依赖于P和L，而转录也需要M2-1。M2-1在重叠结合位点与P和RNA相互作用;虽然这些相互作用是转录活性所必需的，但M2-1作用的机制尚不清楚。为了更好地理解HRSV转录，我们解决了具有最小P相互作用结构域的复合物中M2-1的晶体结构，揭示了M2-1/P界面的分子细节并定义了M2-1在三重复合物中的取向。M2-1/P相互作用相对较弱，这表明高亲和力RNA可以从复合物中取代M2-1，为描述M2-1在转录中的作用的新模型提供了基础。最近，小分子槲皮素和环巴胺已被用于验证M2-1作为药物靶标。人呼吸道合胞病毒（HRSV）是一种负链RNA病毒，引起全球流行的呼吸道感染，可导致危及生命的疾病，特别是在年轻人，老年人和免疫功能低下的人中。HRSV增殖依赖于病毒编码的RNA依赖性RNA聚合酶（RdRp）对HRSV基因的复制和转录。对于复制，该复合物包含磷蛋白（P）和大蛋白（L），而对于转录，还需要M2-1蛋白。M2-1通过与P相互作用被募集到RdRp，并且还在M2-1表面上的重叠结合位点处与RNA相互作用，使得这些伴侣的结合是相互排斥的。M2-1转录需求的分子基础尚不清楚，P和RNA之间竞争M2-1结合的结果也是如此，这可能是转录机制中的关键步骤。在这里，我们报告了与P相互作用结构域结合的M2-1在2.4 nm处的晶体结构，该结构域包含P残基90至110。P90-110肽是α螺旋的，其在M2-1表面上的位置决定了复合物内三种转录酶组分的方向。的M2-1/P接口包括离子，疏水和氢键相互作用，这些接触复杂的形成的关键贡献进行了评估，使用微型基因组测定。使用荧光各向异性定量M2-1对RNA和P配体的亲和力，这表明高亲和力RNA可以胜过P。这对转录机制，特别是转录终止和poly（A）序列合成的事件具有重要意义。
Human respiratory syncytial virus (HRSV) is a leading cause of respiratory illness, particularly in the young, elderly, and immunocompromised, and has also been linked to the development of asthma. HRSV replication depends on P and L, whereas transcription also requires M2-1. M2-1 interacts with P and RNA at overlapping binding sites; while these interactions are necessary for transcriptional activity, the mechanism of M2-1 action is unclear. To better understand HRSV transcription, we solved the crystal structure of M2-1 in complex with the minimal P interaction domain, revealing molecular details of the M2-1/P interface and defining the orientation of M2-1 within the tripartite complex. The M2-1/P interaction is relatively weak, suggesting high-affinity RNAs may displace M2-1 from the complex, providing the basis for a new model describing the role of M2-1 in transcription. Recently, the small molecules quercetin and cyclopamine have been used to validate M2-1 as a drug target. Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause life-threatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90–110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences.