Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors.
Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors.
复制标题
与实验室进化的蛋白结合的HIV TAR RNA的共结构结构显示与与环状肽TAR抑制剂设计有关的精氨酸的关键作用。
DOI：
10.1074/jbc.ra120.015444
复制
发表时间：
2020-12-04
期刊：
影响因子：
--
通讯作者：
Wedekind JE
中科院分区：
文献类型：
--
作者：
Chavali SS;Mali SM;Jenkins JL;Fasan R;Wedekind JE
RNA-蛋白质界面控制着HIV-1生命周期中的关键复制事件。病毒反式转录激活因子(TAT)蛋白利用典型的富含精氨酸的基序(ARM)将宿主阳性转录延伸因子b(PTEFb)复合体招募到病毒反式激活反应(TAR)RNA上，导致HIV转录的激活。阻止这种相互作用的努力刺激了生物制品的生产，这些生物制品旨在破坏这种重要的RNA-蛋白质接口。在这里，我们提出了四种共晶结构的实验室进化的焦油结合蛋白(TBP)与HIV-1TAR的复合体。我们的结果表明，高亲和力结合需要在选择过程中出现的特定β2-β3发夹环中有不同的精氨酸序列和间距。尽管分析了多达5个精氨酸的环，但只有3个精氨酸可以同时与主要沟槽的鸟嘌呤结合。促进β2-β3环内主干相互作用的氨基酸对高亲和力相互作用也是重要的。在结构和亲和力分析的基础上，我们设计了两个模拟焦油结合的β2-β3环序列的环肽，存在于两个高亲和力Tbps中(Kd值分别为4.2±0.3和3.0±0.3 nm)。我们的努力产生了结合具有低微摩尔亲和力(Kd值从3.6到22μm)的焦油的低分子化合物。值得注意的是，在溶液分析中，这一系列中的一个环状化合物阻止了Tat-臂多肽与TAR的结合，而它的线性对应物则没有。总体而言，这项工作为蛋白质介导的焦油识别提供了洞察力，并为开发至关重要的HIV-1RNA-蛋白质相互作用的环肽抑制剂奠定了基础。
RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.