Structural and biochemical characterization of ribosome small subunit-dependent GTPase A (RsgA) from Pseudomonas aeruginosa

The increase in antibiotic resistance among pathogenic bacterial strains presents a significant health threat. The main efforts to combat antibiotic resistance are focused on the development of new antibiotics targeting protein biosynthesis. Ribosome, the large molecular machine responsible for this process, and proteins involved in the translational process represent ideal targets of molecules with antibacterial activity. The ribosome assembly in vivo is an intricate and finely tuned process promoted by the action of several proteins acting as assembly factors, whose precise role is still largely unknown. Small GTPases represent the largest class of ribosome assembly factors in bacteria and are emerging as possible targets to be explored for the development of novel antibacterial strategies. Among them, of particular interest is the Ribosome small subunit-dependent GTPase A (RsgA). RsgA is a late-stage ribosome biogenesis factor involved in the 30S subunit maturation, broadly conserved among bacteria but absent in eukaryotes. RsgA is a circulary permutated GTPase that belongs to an interesting class of GTPases, termed HAS-GTPase, that lack the conserved catalytic glutamine. The circularly permutated GTP binding site is flanked by an OB-fold domain at the N-terminus and by a zinc binding domain at the C-terminus. Despite the large amount of biochemical, structural and genetic data on RsgA achieved in the last decade, its mechanism of action is still not completely understood. Here we focus on the structural and functional characterisation of RsgA from the human pathogenic bacterium Pseudomonas aeruginosa (PaRsgA). The main goal of this work is the determination of the PaRsgA structure by X-ray crystallography. To date, no structure is available for RsgA from this opportunistic pathogen. This knowledge will allow investigate the molecular features for the recognition of GDP and GTP as well as the key determinants for the mechanism of GTP hydrolysis. Moreover, an accurate kinetic analysis of PaRsgA interaction with GDP and GTP, together with a detailed functional characterization of PaRsgA, provided the determination of substrates affinity and biochemical parameters of GTP hydrolysis. The results obtained will pave the way for future experiments aimed at the characterization of the binding mechanism underlying ribosome recognition and to get key insight the GTPase activity of PaRsgA in the presence of other assembly factors and/or the ribosomal particle.