Insights into functionally important conformational changes in the 400 kDa RNA exosome complex: a combination of methyl TROSY NMR spectroscopy and cryo- EM.

蛋白质通常是高度动态的，并根据它们所处的状态改变形状。这些构象变化对于催化循环与蛋白质动力学相关的酶尤其相关。然而，我们目前对大分子机器的了解往往仅限于这些复合物的静态快照。在这里，我们建议量化蛋白质运动和动态基板的相互作用，在真核外泌体复合物。这种重要的分子机器在大量RNA的降解和加工中起着核心作用。酶复合物的催化活性形式包含最少10种不同的蛋白质链（Exo-10），其组装成400 kDa分子机器。已报道了Exo-10复合物的两种结构上不同的构象，已发表的数据表明，这些构象与不同的RNA降解模式相关，因此具有不同的生物学功能。为了直接将Exo-10复合物中的动力学和功能联系起来，我们将在这里使用两种互补的结构生物学方法。在该提案的第一部分中，将利用最先进的甲基TROSY溶液状态NMR技术，以残留物特定的空间分辨率量化Exo-10复合物中的快速和慢速动力学。我们特别感兴趣的是由不同的RNA底物诱导的结构变化。完全不对称的Exo-10复合物是使用溶液状态NMR方法研究过的最具挑战性的复合物之一，我们的初步数据表明，我们确实能够获得位点特异性运动和RNA相互作用。这表明，我们将能够建立大型和完全不对称的真核分子机器是适合详细的溶液状态NMR研究，从而推动技术的限制显着。在提案的第二部分中，我们将使用冷冻EM方法来确定Exo-10复合物在多种不同RNA底物存在下所采用的结构。这些Exo-10复合物的静态快照是准确解释我们的NMR数据所必需的，它们将能够准确地可视化RNA底物朝向活性位点的路线。初步数据表明，我们确实能够获得足够质量的冷冻EM地图。该项目的这两个部分将为我们提供一个大型蛋白质复合物的丰富的NMR和cryo-EM数据。这使我们处于一种独特的情况下，在这种情况下，我们可以解决NMR量化的分子运动在冷冻EM数据中可见的程度。这些见解将是一个普遍的兴趣，因为它可能揭示了必要性，以补充cryo-EM数据与解决方案为基础的方法，以充分解开分子机制。简而言之，我们的数据将提供对中央RNA外泌体复合物的功能见解，同时进一步发展结构生物学方法，以适应日益复杂的系统。

Proteins are often highly dynamic and change shape depending on the state they are in. These conformational changes are especially relevant for enzymes where the catalytic cycle is linked with protein dynamics. Our current knowledge of large molecular machines is, however, often limited to static snapshots of these complexes. Here, we propose to quantify protein motions and dynamic substrate interactions in the eukaryotic exosome complex. This essential molecular machine plays a central role in the degradation and processing of a large number of RNAs. The catalytically active form of the enzyme complex comprises a minimal of 10 different protein chains (Exo-10) that assemble into a 400 kDa molecular machine. Two structurally distinct conformations of the Exo-10 complex have been reported and published data suggest that these are linked with different modes of RNA degradation and thus with different biological functions. To directly relate dynamics and function in the Exo-10 complex we will here make use of two complementary structural biology methods. In the first part of the proposal will exploit state-of- the-art methyl TROSY solution state NMR techniques to quantify fast and slow dynamics in the Exo-10 complex with residue specific spatial resolution. We are especially interested in structural changes that are induced by different RNA substrates. The fully asymmetric Exo-10 complex is one of the most challenging complex that has ever been studied using solution state NMR methods and our preliminary data shows that we are able indeed able to pick up site specific motions and RNA interactions. This illustrates that we will be able to establish that large and fully asymmetric eukaryotic molecular machines are amenable to detailed solution state NMR studies, thereby pushing the limits of the technique significantly. In the second part of the proposal, we will use cryo-EM methods to determine the structures that the Exo-10 complex adopts in the presence of a variety of different RNA substrates. These static snapshots of the Exo-10 complex are required for an accurate interpretation of our NMR data and they will be able to visualize exactly which routes RNA substrates take towards the active sites. Preliminary data shows that we are indeed able to obtain cryo-EM maps of sufficient quality. These two parts of the project will provide us with ample NMR and cryo-EM data for one large protein complex. This puts us then in a unique situation, where we can address to what degree NMR quantified molecular motions are visible in cryo-EM data. These insights will be of a general interest as it might reveal the necessity to complement cryo-EM data with solution based methods to fully unravel molecular mechanisms. In brief, our data will provide functional insights into the central RNA exosome complex and, at the same time, further develops structural biology methodology towards systems of increasing complexity.