Abstract
Efficient adaptation to environmental changes is pivotal for all bacterial cells. Almost all bacterial species depend on the conserved stringent response system to prompt timely transcriptional and metabolic responses according to stress conditions and nutrient depletion. The stringent response relies on the stress-dependent synthesis of the second messenger nucleotides and alarmones (p)ppGpp, which pleiotropically target and reprogram processes that consume cellular resources, such as ribosome biogenesis. Here we show that (p)ppGpp acts on the ribosome biogenesis GTPase A (RbgA) of Gram-positive bacteria. Using X-ray crystallography, hydrogen-deuterium exchange MS (HDX-MS) and kinetic analysis, we demonstrate that the alarmones (p)ppGpp bind to RbgA in a manner similar to that of binding by GDP and GTP and thereby act as competitive inhibitors. Our structural analysis of Staphylococcus aureus RbgA bound to ppGpp and pppGpp at 1.8 and 1.65 Å resolution, respectively, suggested that the alarmones (p)ppGpp prevent the active GTPase conformation of RbgA by sterically blocking the association of its G2 motif via their 3'-pyrophosphate moieties. Taken together, our structural and biochemical characterization of RbgA in the context of the alarmone-mediated stringent response reveals how (p)ppGpp affects the function of RbgA and reprograms this GTPase to arrest the ribosomal large subunit.
Highlights
Efficient adaptation to environmental changes is pivotal for all bacterial cells
To delineate nucleotide-dependent conformational changes of ribosome biogenesis GTPase A (RbgA), we determined crystal structures of S. aureus RbgA bound to GDP and the nonhydrolysable GTP analogue GMPPNP3 at 2.15 and 1.93 Å resolution, respectively (Table 1)
The analysis showed that RbgA adopts different conformations in solution depending on the identity of the nucleotide, which is in contrast to the virtually identical conformation observed in the crystal structures and suggests that crystal packing might have influenced the conformation observed in our structures
Summary
Data collection Space group Cell dimensions a (Å) b (Å) c (Å) ␣ (°)  (°) ␥ (°) Energy (Å) Resolution (Å) No unique reflections Rmerge I/I Completeness (%) Redundancy CC1⁄2. In Saccharomyces cerevisiae, the eukaryotic RbgA homologue Lsg acts late during the final ribosomal large subunit maturation and was shown to be involved in the GTPase-dependent release of the nuclear export adapter Nmd upon 60S subunit completion [15,16,17]. Crystal structures of guanosine nucleotide-bound RbgA homologues of Thermotoga maritima [18] and B. subtilis (PDB code 1PUJ) are available and reveal the N-terminal GTPase fold followed by an ␣-helical C-terminal putative RNA-binding domain. The mechanism by which the stringent response alarmone (p)ppGpp blocks the GTPase activation of RbgA to arrest the maturation of large ribosomal subunits is unknown. Our structural and biochemical analyses of RbgA reveal how the GTPase active conformation is suppressed by (p)ppGpp to arrest large ribosomal subunits during the stringent response
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