The development of NMR methods for drug discovery targeting on ribosome recycling.

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We established NMR assignments of RRFs originated from five bacteria. Resulting assignments bring not only the basis of following structural study, but also the set of interaction probes at an atomic resolution for drug discovery. We investigated inter-molecular dynamics of RRF by NMR relaxation analyses and nanosecond molecular dynamics simulations. The results revealed characteristic flexibility in inter-domain orientation of RRF molecule experimentally, indicating that a compound bound to hinge region would inhibit RRF function. In order to obtain structural information for CARDD, we carried out an X-ray analysis on RRF from Vibrio parahaemolyticus. To elucidate the ribosome binding site of RRF, the peptide fragment corresponding to domain I of RRF (RRF-DI) was expressed and characterized. RRF-DI is bound to 70S ribosome and the 50S subunit with an affinity similar to that of wild-type RRF. But it does not bind to the 30S subunit. Moreover, we carried out a cryo-EM analyses and chemical probing experiments on RRF-ribosome complex. By the virtual screening based on the determined structure and interaction mode of RRF, some candidates for RRF inhibitor were obtained. To assay such compounds, novel screening system in vitro was developed.