Sound the (Smaller) Alarm: The Triphosphate Magic Spot Nucleotide pGpp

Abstract

It has recently become evident that the bacterial stringent response is regulated by a triphosphate alarmone (pGpp) as well as the canonical tetra- and pentaphosphate alarmones ppGpp and pppGpp [together, (p)ppGpp]. Often dismissed in the past as an artifact or degradation product, pGpp has been confirmed as a deliberate endpoint of multiple synthetic pathways utilizing GMP, (p)ppGpp, or GDP/GTP as precursors. Some early studies concluded that pGpp functionally mimics (p)ppGpp and that its biological role is to make alarmone metabolism less dependent on the guanine energy charge of the cell by allowing GMP-dependent synthesis to continue when GDP/GTP has been depleted. However, recent reports that pGpp binds unique potential protein receptors and is the only alarmone synthesized by the intestinal pathogen Clostridioides difficile indicate that pGpp is more than a stand-in for the longer alarmones and plays a distinct biological role beyond its functional overlap (p)ppGpp.

Keywords: (p)ppGpp; alarmone; bacterial nucleotide signaling; pGpp; stringent response.

FIG 1

Structures and synthesis of guanosine alarmones. Shown are the three guanoine alarmones, pppGpp, ppGpp, and pGpp. Alarmone synthesis from GTP/GDP/GMP or (p)ppGpp precursors is catalyzed by synthetase enzymes and alarmone hydrolysis, and modification is catalyzed by hydrolase enzymes. Direct synthesis of pGpp from GTP/GDP in Clostridium difficile is omitted for clarity.

FIG 2

Alarmones complexed with effectors show great diversity in their 5′ phosphate groups. Alarmones and the residues they contact within their protein or RNA binding pockets are pictured. Protein and RNA residues are shown in gray. Hydrogen bonds and salt-bridges are shown as dotted lines. (A) Crystal structure of ppGpp bound to B. subtilis Obg (PDB: 1LNZ). The 5′-disphosphate is completely encased in a binding pocket (yellow circle) that appears sterically capable of accommodating a triphosphate, while the 3′-diphosphate is solvent exposed. This protein binds (p)ppGpp but not pGpp in vitro (33). The exclusion of pGpp is likely due to the direct and magnesium-mediated contacts between the protein and the 5′-β-phosphate. (B) Crystal structure of ppGpp bound to Francisella tularensis MglA-SspA (PDB: 5U51). The 5′-diphosphate contacts the surface of the protein but is solvent-exposed. (C) Crystal structure of pppGpp bound to E. coli PpnN (PDB: 6GFM). The 5′-triphosphate extends out of the binding pocket and has no protein contacts. This protein is pulled out of E. coli lysate by affinity-tagged pppGpp, ppGpp, or pGpp and does not appear to discriminate based on 5′-phosphate group size (31). (D) Crystal structure of ppGpp bound to the Sulfobacillus acidophilus ppGpp riboswitch (PDB: 6DME). The 5′-diphosphate is enveloped in a binding pocket and makes numerous direct contacts with the riboswitch. The scale bar in each image represents 3 Å. Contact maps were generated with LigPlot+.