The detailed characterization of biomolecular interactions requires the combination of different biophysical methods. The concept of integrative structural biology will be illustrated by the structural and functional study of ubiquitous RNase P enzymes, which catalyze the 5’ maturation of pre-tRNAs. For a long time it has been thought that all RNase P were ribozymes. However, a novel kind of RNase P composed of proteins only, called PRORP for “Protein-only RNase P” was first discovered in human mitochondria, then described in Arabidopsis thaliana (1,2). The latter possesses three PRORP homologs: PRORP1 located in mitochondria and chloroplasts, PRORP2 and PRORP3 in the nucleus.
We developed an integrative approach in order to gain an insight into Arabidopsis PRORP enzymes and their mode of action (3). The affinity constant between a minimal tRNA substrate and a catalytically inactive PRORP2 enzyme was first determined by microscale thermophoresis (MST), ultracentrifugation and calorimetry (ITC), and shown to be in the micromolar range. A combination of mutagenesis and affinity measurements helped define the respective importance of individual pentatricopeptide repeats (PPR) of PRORP2 for RNA binding. A comparison of the crystal structure of PRORP2 and of solution structures of the enzyme and its complex with a pre-tRNA obtained by small angle X-ray scattering (SAXS) indicated that PRORP2 undergoes structural changes to accommodate its substrate. A dedicated SAXS setup was implemented to stabilize the complex during analysis. Altogether this study reveals the structural diversity and plasticity of protein-only RNase P enzymes.