The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.
The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.
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DOI：
10.1371/journal.ppat.1002764
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Escalante CR
中科院分区：
文献类型：
--
作者：
Zarate-Perez F;Bardelli M;Burgner JW 2nd;Villamil-Jarauta M;Das K;Kekilli D;Mansilla-Soto J;Linden RM;Escalante CR
腺相关病毒(AAV)的四个Rep蛋白协调病毒生命周期的各个方面，包括转录调控、DNA复制、病毒组装以及病毒基因组与人类19号染色体的定点整合。所有的Rep蛋白都共享一个中心的SF3超家族解旋酶结构域。在其他SF3成员中，这个结构域足以诱导寡聚。然而，AAV Rep蛋白中的解旋酶结构域(即Rep40/Rep52)的单体特性表明，它不能介导稳定的寡聚作用。这一观察结果使我们假设存在一个尚未确定的结构决定因素，它调节Rep齐聚。在这篇文献中，我们描述了AAV-2 Rep蛋白和其他SF3成员的解旋酶结构域之间的详细结构比较。这一分析表明，主要的结构差异存在于Rep解旋酶结构域的小寡聚亚域(OD)。此外，连接解旋酶结构域和起始结合结构域的连接子的二级结构预测表明有可能形成α-螺旋。我们证明了含有不同长度连接子的突变Rep40结构能够形成二聚体，并且在ATP/ADP存在的情况下，能够形成更大的寡聚体。我们进一步鉴定了一个芳香族连接残基(Y224)，它是寡聚的关键，在SF3解旋酶中是一个保守的特征基序。该残基的突变严重影响齐聚反应，并完全丧失产生传染性病毒的能力。综上所述，我们的数据支持这样一个模型，即解旋酶结构域之前的连接子残基折叠成α-螺旋，成为解旋酶结构域的组成部分，并通过与OBD域和解旋酶结构域的协同作用对Rep68/78蛋白的寡聚和功能至关重要。病毒必须优化其有限的基因组大小，以便产生感染和复制所需的蛋白质。有几种机制被用来实现这一点，包括使用多个启动子和选择性剪接。这些过程通过少量蛋白质结构域的组合组装产生具有不同功能的基因产品。腺相关病毒的小基因组有两个主要的开放阅读框架，产生七种蛋白质，四种非结构Rep蛋白和三种衣壳蛋白。非结构Rep蛋白共享一个运动域，该运动域利用ATP的水解来产生构象变化，从而驱动DNA复制、转录调节、位点特异性整合和病毒基因组的衣壳包装。这些功能依赖于特定DNA位点上的Rep蛋白通过N端起始结合域和C端解旋酶结构域的协同作用而发生的寡聚。我们提供的证据表明，连接这两个结构域的连接子是解旋酶结构域的一个完整特征，并且包含一个保守的芳香族残基，这对齐聚是关键的。这个残基是SF3解旋酶的标志性基序，也存在于支持滚环复制机制的细菌Rep蛋白的子集中。
The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains. Viruses have to optimize the limited size of their genomes in order to generate the proteins required for infection and replication. Several mechanisms are used to accomplish this including the use of multiple promoters and alternative splicing. These processes generate gene products with diverse functions through the combinatorial assembly of a small number of protein domains. The small genome of the adeno-associated virus has two major open reading frames that generate seven proteins, four non-structural Rep proteins and three capsid proteins. The non-structural Rep proteins share a motor domain that uses hydrolysis of ATP to generate the conformational changes that drive DNA replication, transcriptional regulation, site-specific integration and the packing of viral genome into capsids. These functions depend upon the oligomerization of Rep proteins on specific DNA sites through the cooperation of the N-terminal origin binding domain and the C-terminal helicase domain. We provide evidence that the linker that connects the two domains is an integral feature of the helicase domain and contains a conserved aromatic residue that is critical for oligomerization. This residue emerges to be a signature motif of SF3 helicases and is also present in a subset of bacterial Rep proteins that support rolling circle replication mechanism.
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