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.

中文翻译：

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HIV TAR RNA 与实验室进化蛋白结合的共晶结构显示精氨酸在环肽 TAR 抑制剂设计中的关键作用

RNA-蛋白质界面控制 HIV-1 生命周期中的关键复制事件。病毒转录反式激活蛋白 (Tat) 使用典型的富含精氨酸基序 (ARM) 将宿主正转录延伸因子 b (pTEFb) 复合物募集到病毒反式激活反应 (TAR) RNA 上，从而激活HIV转录。阻止这种相互作用的努力刺激了旨在破坏这种重要的 RNA-蛋白质界面的生物制剂的生产。在这里，我们展示了实验室进化的 TAR 结合蛋白 (TBP) 与 HIV-1 TAR 复合物的四种共晶结构。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 发夹环内具有独特的精氨酸序列和间距。尽管分析了多达五个精氨酸的环，只有三个精氨酸可以同时与大沟鸟嘌呤结合。还观察到促进 β2-β3 环内主链相互作用的氨基酸对于高亲和力相互作用很重要。基于结构和亲和力分析，我们设计了两个高亲和力 TBP 中存在的 TAR 结合 β2-β3 环序列的两个环肽模拟物（KD 值分别为 4.2 ± 0.3 和 3.0 ± 0.3 nm）。我们的努力产生了低分子量化合物，以低微摩尔亲和力（KD 值范围为 3.6 至 22 μm）结合 TAR。值得注意的是，该系列中的一种环状化合物在溶液测定中阻断了 Tat-ARM 肽与 TAR 的结合，而其线性对应物则不然。全面的，