Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy
Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy
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DOI：
10.1021/bi992932p
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发表时间：
2000-05-16
期刊：
影响因子：
2.9
通讯作者：
Marino, JP
中科院分区：
文献类型：
--
作者：
Lacourciere, KA;Stivers, JT;Marino, JP
Rev是一种必需的HIV-1调节蛋白，其结合HIV-1 RNA基因组的env基因内的Rev反应元件（RRE），并参与未剪接或部分剪接的病毒mRNA从细胞核转运至细胞质。先前的研究已经表明，源自Rev（Rev 34-50）的短α-螺旋肽和RRE序列的截短形式提供了有用的体外系统来研究这种相互作用，同时仍然保留天然复合物的基本方面。我们选择性地将荧光探针2-氨基嘌呤2 '-O-甲基核苷（2-AP）掺入RRE序列的非干扰位置（A68和U 72），从而可以通过荧光方法直接表征Rev肽和氨基糖苷配体的结合。Rev肽与RRE-72 AP变体的结合导致荧光增加2倍，这为监测这种结合相互作用提供了有用的信号（K-D = 20 +/- 7 nM）。使用停流动力学测量，我们已经表明，特定的Rev肽结合发生的两步过程，涉及扩散控制的遭遇，然后通过异构化的RNA。使用RRE-68 AP和-72AP构建体，明确检测到氨基糖苷类新霉素的三类结合位点。第一个位点对Rev结合无抑制作用（K-D = 0.24 +/- 0.040 μ M），第二个位点以竞争方式抑制Rev结合（K-D = 1.8 +/- 0.8 μ M），第三个弱得多的位点（或多个位点）归因于非特异性结合（K-D大于或等于40 μ M）。互补的NMR测量结果表明，新霉素形成一个特定的二元复合物与RRE和特定的三元复合物与RRE和Rev. NMR数据进一步表明，新霉素占据了一个类似的高亲和力结合位点的二元和三元复合物，这个网站是位于较低的茎区域的RRE。
Rev is an essential HIV-I regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short a-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K-D = 20 +/- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K-D = 0.24 +/- 0.040 mu M), the second site inhibited Rev binding in a competitive fashion (K-D = 1.8 +/- 0.8 mu M), and the third much weaker site (or sites) is attributed to nonspecific binding (K-D greater than or equal to 40 mu M). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and tertiary complexes, and that this site is located in the lower stem region of RRE.