Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.
Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.
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DOI：
10.1021/acs.biochem.5b00610
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发表时间：
2015-09-29
期刊：
影响因子：
2.9
通讯作者：
Escalante CR
中科院分区：
文献类型：
--
作者：
Musayev FN;Zarate-Perez F;Bardelli M;Bishop C;Saniev EF;Linden RM;Henckaerts E;Escalante CR
腺相关病毒(AAV)非结构蛋白Rep78和Rep68执行调节AAV生命周期的所有DNA交易。它们共享两个多功能结构域：来自HH超家族的N末端起始结合/缺口结构域(OBD)和SF3解旋酶结构域。一个由~20个氨基酸组成的短链连接着这两个结构域，它对齐聚和功能至关重要。虽然AAV5OBD和AAV2解旋酶结构域的X射线结构已经确定，但关于全长蛋白质和连接子构象的信息尚不清楚。本文介绍了AAV2Rep68的小角X射线散射(SAXS)溶液结构。我们首先确定了最小的AAV2Rep68OBD和带有连接区的OBD的X射线结构。这些X射线结构揭示了新的特征，包括从蛋白质核心以45°角伸出的长C-末端α-螺旋和部分结构化接头。SAXS研究证实了连接体没有延伸，我们表明连接体中的Pro残基对Rep68的齐聚和功能是关键的。基于SAXS的Rep68刚体建模证实了这些观察结果，显示了两个结构域的紧密排列，其中它们获得了一种构象，将所有结构域中的关键残基定位在蛋白质的一个面上，准备与DNA相互作用。
Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ~20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.