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

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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.