Functional dynamics of the eukaryotic RNA exosome upon complex formation

Abstract

Rather than being static, proteins adopt multiple conformations and undergo dynamics on a wide range of timescales. Such conformational changes can be routinely probed by solution state NMR for protein sizes below 40 kDa and can be extended to large protein complexes with selective methyl-labeling strategies. Here, we expand the limit of methyl-NMR dynamics experiments to asymmetric protein complexes bigger than 300 kDa by investigating the inert core of the eukaryotic RNA exosome, Exo9. We demonstrate that isolated subunits are highly dynamic on the ms-s dynamics, which facilitates assembly. While ring subunits lose dynamics upon complex-formation, cap subunits and functional loops retain a large degree of flexibility as demonstrated by relaxation dispersion and chemical exchange saturation transfer experiments in the fully asymmetric Exo9 complex. Employing EPR spectroscopy we additionally revealed changes in the conformational ensemble and nanosecond dynamics of a spin-labeled functional loop region in the presence of the catalytic subunit Rrp44 and in response to substrate RNA binding. We thus demonstrate that an integrated NMR-EPR approach facilitates the characterization of functional dynamics that occur on a wide range of timescales in very large protein complexes.