RNase P is a ubiquitous and essential ribonucleoprotein complex, responsible for 5' leader sequence removal during tRNA maturation. It is the RNA component that performs the catalysis, making it a ribozyme. Bacterial RNase P is best understood. The structures of both the RNA and the single protein cofactor have been determined by X-ray crystallography and NMR spectroscopy. Thorough biochemical studies have revealed how the bacterial RNA subunit appropriately positions catalytic Mg2+ ions to catalyze water-mediated nucleophilic attack on the phosphodiester bond, and how the protein subunit assists the RNA structurally. On the contrary, eukaryotic RNase P has been quite a challenge to work with because of its complexity (one RNA and 9 to 10 proteins) and the lack of a robust reconstitution assay for functional studies. Because of evolutionary divergence, the results from bacterial RNase P studies can not be readily transferred to understanding the eukaryal counterpart. Archaeal RNase P appears to be an intermediate between bacterial and eukaryotic RNase P. It is composed of one RNA and 5 protein subunits, each of which has a eukaryotic homolog. Four of them function in pairs (RPP21-RPP29 and RPP30-POP5). Importantly, the resemblance between archaeal and eukayotic RNase P regarding their sequences and interaction implies that the knowledge gained from the simpler archaeal RNase P can be reasonably projected onto the eukaryotic enzyme. We used solution NMR spectroscopy to determine the structure of the protein-protein complex comprising Pyrococcus furiosus RPP29 and RPP21. We found that the protein-protein interaction is characterized by coupled folding of secondary structural elements that participate in interface formation. In addition to detailing the intermolecular contacts that stabilize this 30-kDa binary complex, the structure identifies a surface rich in conserved basic residues likely vital for recognition of the RNA subunit and / or precursor tRNA. These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a 3D model of this ancient RNP enzyme.
Binding-coupled protein folding observed in both RPP29 and RPP21 directed our research into thermodynamic characterization of this binary complex using ITC. These experiments revealed a large excess ΔCp in support of the NMR-observed induced fit occurring during complex formation. To quantitatively correlate the ΔCp to the extent of folding upon binding, we investigated the ion and proton linkage effects which can also contribute to ΔCp. Interestingly, though two salt bridges have been proposed based on the structure, high salt concentration was not seen to impair the RPP21-RPP29 interaction. Instead, high salt concentration resulted in tighter binding. In addition, we found that this ion effect does not contribute significantly to ΔCp. ITC experiments performed in the buffers with different ionization enthalpy indicated that two protons are released upon binding. Using an empirically determined method, we find that the corrected ΔCp reports 50 residues that fold during complex formation, consistent with the extent of folding observed in the NMR studies.