Structural and functional investigation of the Small Ribosomal Subunit Biogenesis GTPase A (RsgA) from Pseudomonas aeruginosa

Abstract

The Small Ribosomal Subunit Biogenesis GTPase A (RsgA) is a bacterial assembly factor involved in the late stages of the 30S subunit maturation. It is a multidomain GTPase in which the central circularly permutated GTPase domain is flanked by an OB domain and a Zn‐binding domain. All three domains participate in the interaction with the 30S particle thus ensuring an efficient coupling between catalytic activity and biological function. In vivo studies suggested the relevance of rsgA in bacterial growth and cellular viability, but other pleiotropic roles of RsgA are also emerging. Here, we report the 3D structure of RsgA from Pseudomonas aeruginosa (PaRsgA) in the GDP‐bound form. We also report a biophysical and biochemical characterization of the protein in both the GDP‐bound and its nucleotide‐free form. In particular, we report a kinetic analysis of the RsgA binding to GTP and GDP. We found that PaRsgA is able to bind both nucleotides with submicromolar affinity. The higher affinity towards GDP (KD = 0.011 μm) with respect to GTP (KD = 0.16 μm) is mainly ascribed to a smaller GDP dissociation rate. Our results confirm that PaRsgA, like most other GTPases, has a weak intrinsic enzymatic activity (kCAT = 0.058 min−1). Finally, the biological role of RsgA in P. aeruginosa was investigated, allowing us to conclude that rsgA is dispensable for P. aeruginosa growth but important for drug resistance and virulence in an animal infection model. Databases Coordinates and structure factors for the protein structure described in this manuscript have been deposited in the Protein Data Bank (http://www.rcsb.org/pdb) with the accession code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6H4D.

Genetic Basis and Physiological Effects of Lipid A Hydroxylation in Pseudomonas aeruginosa PAO1

“…Bacterial strains used in this study are listed in Table S1. Bacteria were cultured in Lysogeny Broth, Lennox formulation (Acumedia) for genetic manipulation, while growth, biofilm, and antibiotic resistance assays were performed in Mueller-Hinton broth (MH; Difco) or M9 minimal medium [17] supplemented with 50 µM FeCl 3 and 20 mM succinate as the carbon source. Planktonic growth assays were performed in 96-well microtiter plates (200 µL of medium in each well) at 22, 30, or 37 • C and the optical density at 600 nm (OD 600 ) of bacterial cultures was measured in a Tecan Spark 10M microtiter reader.…”

The Impact of the Stringent Response on TRAFAC GTPases and Prokaryotic Ribosome Assembly

“…The multidomain GTPase RsgA (YloQ in B. subtilis , YjeQ in E. coli ) is a late-stage 30S ribosome assembly cofactor that is widely conserved among bacteria, although is not essential for growth [6,51]. It contains the conserved G-motifs arranged in an alternative G4-G5-G1-G2-G3 permutation (Figure 2), making it a member of the circularly-permuted GTPase family (CP-GTPase) alongside the other ribosome biogenesis cofactors YawG, YqeH and RbgA.…”

Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria

“…YjeQ (about 33 kDa in Thermotoga maritima) consists of three domains (Figure 3b) [39][40][41][42]. The N-terminal or OB-fold domain (oligonucleotide/oligosaccharide-binding) represents a β-barrel.…”

“…N-terminal amino acid residues from 1 to 20 are essential for tight binding to the 30S ribosomal subunit [33]. The central GTPase domain is a Rossmann fold with a unique circular permutation: instead of the standard order G1-G2-G3-G4-G5, which is characteristic of TRAFAC (TRAnslation FACtor) GTPases, it possesses a G4-G5-G1-G2-G3 motif because the N-terminal and C-terminal loops are swapped [39][40][41][42]. As a result of this rearrangement, the C-terminal domain may have a direct mechanical effect on the GTP hydrolysis in YjeQ.…”

“…The C-terminal part consists of one 3 10 -helix and the two α-helices with the zinc-finger motif Cx(F/Y)xxCxHxx(E/D)xxC. [39][40][41][42]. These cysteines and histidine are located in the loop connecting two α-helices and coordinate a zinc ion.…”