Interactions between HIV-1 Gag molecules in solution: An inositol phosphate-mediated switch
Interactions between HIV-1 Gag molecules in solution: An inositol phosphate-mediated switch
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DOI：
10.1016/j.jmb.2006.10.072
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发表时间：
2007-01-19
影响因子：
5.6
通讯作者：
Rein, Alan
中科院分区：
文献类型：
--
作者：
Datta, Siddhartha A. K.;Zhao, Zhuojun;Rein, Alan
逆转录病毒颗粒组装是由Gag蛋白介导的。GAG是一种含有由柔性连接子连接的离散结构域的多结构域蛋白。重组HIV-1 Gag蛋白(N端缺失肉豆蔻酸，C端缺失p6结构域)与核酸混合后，在体外形成病毒样颗粒(VLP)。然而，这种组装是有缺陷的，因为VLP的曲率半径远远小于真正的未成熟病毒粒子的曲率半径。这一缺陷可以通过在组装反应中添加肌醇磷酸盐来不同程度地纠正。我们现在已经探索了六磷酸肌醇(IP6)与GAG的结合以及它对溶液中GAG蛋白分子相互作用的影响。我们的数据表明，蛋白质两端的碱性区域有助于IP6的结合。GAG在溶液中处于单体-二聚体平衡状态，其衣壳结构域中二聚体界面的突变极大地减少了GAG的二聚化。相反，当加入IP6时，GAG处于单体-三聚体平衡状态，而不是单体-二聚体平衡状态。在IP6中，二聚体界面突变的Gag蛋白也几乎保持为单体；因此，“二聚体界面”对于IP6中的三聚体相互作用是必不可少的。我们讨论了这些结果的可能解释，包括IP6与蛋白质内其他结构域结合引起的衣壳结构域内构象的变化。GAG的两端都参与了IP6的相互作用，这表明GAG在溶液中是折叠的，其末端在三维空间中相邻；在配套的手稿中为这一结论提供了直接支持。由于Gag是未成熟病毒粒子中的延伸杆，因此在溶液中末端的明显接近意味着它在粒子组装过程中经历了主要的构象变化。(C)2006爱思唯尔有限公司。保留所有权利。
Retrovirus particle assembly is mediated by the Gag protein. Gag is a multidomain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly. (c) 2006 Elsevier Ltd. All rights reserved.