Conformational and Functional Divergence of RnpA Protein Subfamilies in Bacterial Ribonucleoprotein RNase P

Bacterial ribonuclease P (RNase P) is an essential ribonucleoprotein enzyme that catalyzes 5' leader removal from precursor tRNAs (ptRNAs) using a catalytic RNA subunit (P RNA) and an essential protein cofactor (RnpA). RnpA binds near the P RNA active site, facilitating catalysis and enhancing ptRNA binding by contacting 5' leader sequences. However, key information regarding how sequence variation, particularly among bacterial pathogens, influences folding, dynamics, P RNA activation, and catalytic function is lacking. Using sequence similarity network (SSN) analyses of >1800 RnpA sequences, we identify two major subfamilies, a Bacilli-specific class (RnpA-1) that associates with divergent Type B P RNAs, and a broader class (RnpA-2) that associates with ancestral Type A P RNAs, with species-specific variation concentrated at the N- and C-termini. Computational and biophysical studies show that RnpA-1 proteins, including those from Staphylococcus aureus and Enterococcus faecium, exhibit greater conformational dynamics and reduced thermal stability relative to RnpA-2 proteins from representative Gram-negative pathogens. Despite these differences, both families comparably enhance binding of ptRNA to their cognate Type A or B P RNA. In contrast, kinetic studies reveal higher kcat values for Type B RNase Ps and rate limiting product release, while Type A enzymes are limited by precatalytic steps. Reconstitution with noncognate subunits produces selective defects in either kcat or KM demonstrating that RnpA identity makes distinct contributions to substrate binding and catalytic activation. These results further define the conserved and divergent structural, dynamic and functional features of RnpA proteins and establish a foundation for understanding biological function and inhibitor targeting.