Structural basis for (p)ppGpp-mediated inhibition of the GTPase RbgA

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.

IM30 IDPs form a membrane protective carpet upon super-complex disassembly

“…Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was essentially conducted as described previously 46,47 . Sample preparation was aided by a two-arm robotic autosampler (LEAP Technologies).…”

Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

“…mt-LAF4 is bound to H33-35 and appears here to prevent uL2m association. Mt-LAF3 (homolog to the bacterial GTPase RgbA 18 ) is in contact with mt-LAF 5 and 2. However, it is the only GTPase that does not make significant contacts with the rRNA (Fig.…”

(p)ppGpp directly regulates translation initiation during entry into quiescence

“…Given these observations, we wished to identify the component(s) of the translation machinery that is (are) targeted by ppGpp. The translational GTPases EF-Tu, EF-G and IF2 as well as the ribosome associated GTPases including Obg (Buglino et al, 2002; Feng et al, 2014), RsgA (Corrigan et al, 2016; Zhang et al, 2018), RbgA (Corrigan et al, 2016; Pausch et al, 2018), Era (Corrigan et al, 2016), and HflX (Corrigan et al, 2016; Zhang et al, 2018) have all been reported to bind (p)ppGpp. However, (p)ppGpp inhibits protein synthesis by the PURExpress system (Figure 4), which contains only IF2, EF-Tu, and EF-G, so inhibition of one or more of these proteins is likely sufficient to account for the in vivo inhibitory effect of (p)ppGpp on translation.…”

“…We show that the (p)ppGpp sensitivity of IF2 can be altered by a specific double mutation in the G1 motif of the G domain, the site of (p)ppGpp binding (Milon et al, 2006). There are numerous examples of other ribosome associated GTPases that bind (p)ppGpp such as ObgE (Buglino et al, 2002; Feng et al, 2014; Persky et al, 2009), BipA (Kumar et al, 2015), RbgA (Corrigan et al, 2016; Pausch et al, 2018), HflX (Corrigan et al, 2016) and Era (Corrigan et al, 2016). Many of these reports demonstrate that (p)ppGpp binding affects their in vitro function in aspects of protein synthesis, such as ribosome assembly.…”

“…In vitro , (p)ppGpp inhibits translation in a manner similar to that observed with non- hydrolyzable GTP analogs (Wagner and Kurland, 1980), suggesting that (p)ppGpp is targeting a translational GTPase. Consistently, (p)ppGpp inhibits the GTPase activity of IF2 and EF-Tu (Hamel and Cashel, 1974) as well as of GTPases that mediate other aspects of translation such as ribosome assembly (Corrigan et al, 2016; Pausch et al, 2018), by acting as competitive inhibitors. (p)ppGpp is capable of binding translational GTPases including EF-Tu, EF-G (Rojas et al, 1984), IF2 (Milon et al, 2006; Mitkevich et al, 2010) and the ribosome assembly GTPase ObgE (Persky et al, 2009) at affinities that are commensurate with the in vivo levels of (p)ppGpp observed following stringent response induction, consistent with the enzymes being in vivo targets.…”