Direct structural evidence supporting a revolving mechanism in DNA packaging motors.
Direct structural evidence supporting a revolving mechanism in DNA packaging motors.
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支持 DNA 包装电机旋转机制的直接结构证据
DOI：
10.1007/s41048-020-00115-w
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发表时间：
2020-10-31
影响因子：
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通讯作者：
Cheng, Xiaolin
中科院分区：
文献类型：
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作者：
Shu, Yao-Gen;Cheng, Xiaolin
环是由三种蛋白质(pUL15、pUL28和pUL33)相互交错形成的杂三聚体。终止酶pUL15折叠成‘L’型结构，包含5个功能结构域：N-套索(残基1-152)、支链(残基153-252)、三磷酸腺苷酶(残基253-413)、调节子(残基414-478)和核酸酶(残基479-735)。结构分析也模糊地确定R346是反式作用的精氨酸手指，它从ATPase亚基延伸到其邻近的ATP结合口袋与磷酸酯相互作用。2008)和SF6 GP2(赵等人2013)，疱疹病毒pUL15是唯一被确定为具有潜在功能的寡聚体状态的TerL结构。因此，pUL15结构为理解ATPase马达相对于其DNA底物的相对取向以及单个亚基在其寡聚体状态下如何相互作用提供了重要的模板。我们以它们的ATPase结构域为参照，对三个单体TerL结构进行了简单的结构叠加。我们重点研究了大多数DNA包装马达共有的三个结构域，即ATPase结构域(对应于/29 gp16的N-末端结构域，这里称为NTD)、调节结构域(对应于/29 gp16的连接器域)和核酸酶结构域(对应于/29 gp16的C-末端结构域，这里称为CTD)。如图1所示，尽管它们在病毒家族中有较远的亲缘关系，但所有三个ATPase结构域都非常好地比对，同时观察到各个结构域的空间排列有很大的差异。NTD-CTD的分离和CTD相对于NTD的取向在三个Terl中都不同。在疱疹病毒pUL15中，NTD和CTD没有直接接触，而T4 gp17和SF6 gp2两个结构域之间存在广泛的相互作用。另一个有趣的观察是，pUL15中核酸酶结构域的催化部位向邻近的亚基开放，而不是DNA，这表明核酸酶活性在易位过程中受到抑制(Yang等人。2020)。然而，这一信息从T4 gp17或SF6 GP2的单体结构中并不是立即清楚的，但根据排列的结构，很明显，这两个核酸酶位点都不能被DNA访问，T4 gp17和SF6 GP2都需要构象变化才能暴露它们的核酸酶位点，以便在包装时进行DNA切割。
ring is a heterotrimer formed by the three proteins (pUL15, pUL28 and pUL33) interdigitating with each other. The terminase pUL15 folds into an ‘‘L’’shaped structure, containing five functional domains: N-lasso (residues 1–152), strut (residues 153–252), ATPase (residues 253–413), regulator (residues 414–478) and nuclease (residues 479–735). Structural analysis also ambiguously identified R346 as the trans-acting arginine finger that extends from an ATPase subunit to its adjacent ATP binding pocket to interact with the cphosphate.Although atomic structures of several full-length viral large terminase subunits, such as T4 gp17 (Sun et al. 2008) and Sf6 gp2 (Zhao et al. 2013), are available, herpesvirus pUL15 is the only TerL structure determined in a potentially ‘‘functional’’oligomeric state. Therefore, the pUL15 structure provides an important template for understanding the relative orientation of the ATPase motors with respect to their DNA substrates and how the individual subunits interact with each other in their oligomeric state. We performed simple structural superposition of the three monomeric TerL structures using their ATPase domains as a reference. We focused on the three domains common to most DNA packaging motors, the ATPase domain (corresponding to the N-terminal domain of/29 gp16, referred to here as NTD), the regulator domain (corresponding to the linker domain of/29 gp16) and the nuclease domain (corresponding to the C-terminal domain of/29 gp16, referred to here as CTD). As shown in Fig. 1, despite their distant relation in the viral family, all the three ATPase domains are aligned remarkably well while large variations are observed in the spatial arrangements of individual domains. Both the NTD–CTD separation and the orientation of CTD relative to NTD vary among the three TerLs. In herpesvirus pUL15, NTD and CTD make no direct contact with each other while both T4 gp17 and Sf6 gp2 show extensive interactions between the two domains. Another intriguing observation is that the catalytic site of the nuclease domain in pUL15 opens towards the adjacent subunit instead of DNA, suggesting the nuclease activity is inhibited during translocation (Yang et al. 2020). This information is, however, not immediately clear from the monomeric structure of either T4 gp17 or Sf6 gp2, but based on the aligned structures, it is evident that both nuclease sites are inaccessible to DNA, and conformational changes would be required for both T4 gp17 and Sf6 gp2 to expose their nuclease sites for DNA cleavage upon packaging.