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. 2018 Dec 21;293(51):19699-19709.
doi: 10.1074/jbc.RA118.003070. Epub 2018 Oct 26.

Structural Basis for (p)ppGpp-mediated Inhibition of the GTPase RbgA

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Free PMC article

Structural Basis for (p)ppGpp-mediated Inhibition of the GTPase RbgA

Patrick Pausch et al. J Biol Chem. .
Free PMC article

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.

Keywords: (p)ppGpp; GTPase; RbgA; X-ray crystallography; alarmone; cell stress; enzyme kinetics; inhibition mechanism; ribosome assembly; stringent response.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Crystal structures of S. aureus RbgA bound to GDP and GTP. A, amino acid sequence of RbgA illustrating the domain arrangement (N-terminal G domain and C-terminal RNA-binding domain). G motifs are highlighted in turquoise and labeled according to the identity (G1–G5). B and C, crystal structure of RbgA (gray cartoon representation) in complex with GDP (B) and GMPPNP (C) (yellow stick representation) in two 180° rotated views. Secondary structure elements are labeled according to their identity, and N and C indicate the respective termini. The G1–G5 motifs are colored turquoise and are labeled accordingly. D and E, detailed view on the GTPase active sites with the accommodated nucleotide GDP (D) and GMPPNP (E). Coloring is as in B. G motif and adjacent site chains are shown in stick representation and are labeled according to their identity.
Figure 2.
Figure 2.
Crystal structures of S. aureus RbgA bound to ppGpp and pppGpp. A and B, left panels, crystal structure of RbgA (gray cartoon representation) in complex with ppGpp (A) and pppGpp (B) (yellow stick representation). Secondary structure elements are labeled according to their identity, and N indicates the N terminus. The G1–G5 motifs are labeled accordingly. Right panels, close-up on the GTP binding site. Lys-88 (blue stick representation) of the G5 motif is in close proximity to the ϵ-phosphate moiety. C, binding and dissociation constants for GTP, GDP, ppGpp, or pppGpp and RbgA as determined by MST. Error bars represent the standard deviation of the calculated Kd or Ka values based on the fitting of the respective experimental data (Fig. S4).
Figure 3.
Figure 3.
Binding of Mg2+ coincides with rearrangement of the G3 motif (switch II). A, upper panel, overview of the crystal structure of S. aureus RbgA (gray cartoon) in complex with GMPPNP (yellow stick representation). G motifs that participate in phosphate coordination and hydrolysis are colored turquoise and labeled according to their identity. Adjacent helices are labeled according to their identity for orientation (compare with fig. 1). Lower panel, close-up of the GTPase active site. The side chains of the G motifs G1–G3 are shown in blue stick representation. Magenta arrows emphasize the rearrangements that have to occur to locate the G2 motif (switch I; S-I) and G3 motif (switch II; S-II) in a position compatible with GTP hydrolysis. B, crystal structure of T. maritima RbgA (PDB code 3CNN (18)). Representations are as in A. C, crystal structure of B. subtilis RbgA (PDB code 1PUJ). Representations are as in A and B. In contrast to the structures of S. aureus and T. maritima, B. subtilis RbgA assumes a configuration in which the G3 motif is repositioned to allow for Mg2+ (green sphere) coordination.
Figure 4.
Figure 4.
Alarmones bind to the canonical GTP-binding site of RbgA. A, representative peptides of SaRbgA are colored according to their difference in HDX between nucleotide-bound (i.e. GDP, GMPPNP, ppGpp, and pppGpp) SaRbgA and apo-SaRbgA. The positions of the G-elements within the highlighted regions are indicated. B, time courses of deuterium uptake of regions R1–R4 of SaRbgA in the apo- and different nucleotide-bound states. C, location of regions R1–R4 displaying differences in HDX on the crystal structure of SaRbgA bound to pppGpp (this study).
Figure 5.
Figure 5.
RbgA GTPase inhibition by (p)ppGpp. Shown is a comparison of the three different GTPase G1, G2, and G3 motif (turquoise) configurations observed in the crystal structures of S. aureus RbgA (left panel; this study), B. subtilis RbgA (middle panel; PDB code 1PUJ), and cryo-EM structure of S. cerevisiae Lsg1 (right panel; PDB code 5T62 (17)). RbgA/Lsg1 is shown in a gray cartoon representation in a close-up view on the GTPase active site. The associated nucleotides are shown in a yellow stick representation, and the coordinated Mg2+ ion is shown as a green sphere. Rearrangements of switches I and II required for GTPase activation are indicated by magenta arrows. The δ- and ϵ-phosphate moiety of (p)ppGpp sterically blocks the association of the G2 motif as suggested by the G motif configuration observed in the Lsg1 homologue (indicated as an orange oval).
Figure 6.
Figure 6.
Alarmones competitively inhibit the GTPase activity of BsRbgA. A, BsRbgA was incubated with GTP, ppGpp, or pppGpp in the absence (gray bars) or presence (black bars) of 50S subunits, and its hydrolytic activity was determined by HPLC. Error bars indicate standard deviations derived from three individual measurements. B, Lineweaver–Burk plots of BsRbgA GTPase activity without or with purified 50S subunits in the presence of increasing concentrations of ppGpp (left panels) or pppGpp (right panels). The GTP concentration and initial velocity are given in mm−1 and min*nmol RbgA*nmol GTP−1, respectively. C, the initial velocities of GTPase activity of BsRbgA in presence of 1 mm GTP and increasing amounts of ppGpp (red line, squares) or pppGpp (green line, circles). Solid and dashed lines indicate the presence or absence of 50S subunits, respectively. Error bars represent standard deviations, derived from triplicates. The inhibitory constants (Ki) are shown on the right side.
Figure 7.
Figure 7.
Configuration control of YRG-type GTPase G motifs by the relative arrangement of H69/H71 of the large subunit. Shown are the crystal structures of S. aureus (yellow, this study) and B. subtilis (green, PDB code 1PUJ) aligned to the G1 motif of the cryo-EM structure of S. cerevisiae Lsg1 (blue, PDB code 5T62 (17)). The relative position of the flexibly connected N-terminal G domain and C-terminal RNA-binding domain of the YRG-type GTPases depends on the configuration of H69/H71. The mature configuration of H69/H71 might eventually signal for large ribosomal subunit completion to allow for GTPase activation by proper positioning of the G motifs (G1–G5) and subsequent GTPase release.

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