Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology
Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology
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DOI：
10.1007/s10858-012-9696-4
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发表时间：
2013-01-01
影响因子：
2.7
通讯作者：
Allain, Frederic H. -T.
中科院分区：
文献类型：
--
作者：
Barraud, Pierre;Allain, Frederic H. -T.
人hnRNPA 1是一种多功能蛋白，参与选择性剪接、micro-RNA合成、核质mRNA转运、端粒合成和维持等核酸加工过程。hnRNP A1的N-末端区域，也称为解旋蛋白1（UP 1），由两个密切相关的RNA识别基序（RRM）组成，随后是C-末端富含甘氨酸的区域。虽然UP 1的晶体结构揭示了RRM 1和RRM 2之间的域间相互作用，在自由和结合形式的UP 1，这些相互作用从来没有建立在解决方案中。此外，hnRNP A1 RRM的相对取向是不同的自由和绑定晶体结构的UP 1，提出了这个域运动的生物意义的问题。在本研究中，我们使用NMR光谱结合节段性同位素标记技术仔细分析了溶液中存在的RRM间接触，随后确定了溶液中UP 1的结构。我们的数据明确地表明，hnRNP A1 RRM在溶液中相互作用，令人惊讶的是，在溶液中观察到的两个RRM的相对取向是不同的一个发现在晶体结构中的自由UP 1，而类似于一个观察到的核酸结合形式的蛋白质。这强烈支持了hnRNP A1的两个RRM具有单一定义的相对取向的想法，该取向是先前在结合形式中观察到的构象，并且现在使用NMR在溶液中观察到。游离形式的晶体结构中的构象可能是由晶体接触诱导的不太稳定的形式。重要的是，在含有多个RRM的蛋白质中RRM的相对取向强烈影响这些蛋白质实际上可接近的RNA结合拓扑结构。实际上，RRM结构域是以单一限定取向接触单链核酸的不对称结合平台。因此，核酸分子在多个RRM结构域上的路径强烈依赖于RRM是否彼此相互作用。本文在现有结构信息的基础上，对多RRM结构域对核酸的不同识别模式进行了简要的综述和分析。
Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other. The different nucleic acid recognition modes by multiple-RRM domains are briefly reviewed and analyzed on the basis of the current structural information.