Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction

Abstract

The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here, we elucidate the mechanism by which basal (p)ppGpp inhibits the purine salvage enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates with HPRT oligomeric forms. (p)ppGpp-sensitive HPRT exists as a PRPP-bound dimer or an apo- and (p)ppGpp-bound tetramer, where a dimer-dimer interface triggers allosteric structural rearrangements to enhance (p)ppGpp inhibition. Loss of this oligomeric interface results in weakened (p)ppGpp regulation. Our results reveal an evolutionary principle whereby protein oligomerization allows evolutionary change to accumulate away from a conserved binding pocket to allosterically alter specificity of ligand interaction. This principle also explains how another (p)ppGpp target GMK is variably regulated across species. Since most ligands bind near protein interfaces, we propose that this principle extends to many other protein–ligand interactions.

The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp

“…e HprT enzymatic activities in the presence of indicated concentrations of pGpp, ppGpp, and pppGpp. The reaction was performed with 20 nM HPRT, 1 mM PRPP, and 50 μM guanine 14 . Error bars represent standard errors of the mean from three replicates.…”

“…The regulation of purine nucleotide synthesis in firmicutes includes well characterized targets Gmk and HprT whose regulation by (p)ppGpp protects Bacillus subtilis against nutrient changes like amino acid starvation and purine fluctuation, preventing cells from accumulating toxic high levels of intracellular GTP 12 , 14 , 35 . These also include new-found likely targets such as enzymes GuaC and PurA and the de novo pathway transcription factor PurR.…”

Possible Roles for Basal Levels of (p)ppGpp: Growth Efficiency Vs. Surviving Stress

“…After accumulation, (p)ppGpp will change gene expression patterns by binding to RNA polymerase (RNAP) in Gram-negative bacteria or in Gram positives by lowering GTP levels, which is sensed by the transcription factor CodY (reviewed by Gaca et al, 2015a ). Alternatively, (p)ppGpp can directly bind to some proteins and alter their synthetic activities ( Zhang et al, 2018 ; Anderson et al, 2019 ; Wang et al, 2019 , 2020 ).…”

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

“…Conversely, in Gram‐positive bacteria (including Bacillus subtilis ), (p)ppGpp does not target RNAP. Instead, GTP biosynthesis is the primary target of (p)ppGpp (Anderson et al, ; Barker et al, a, b; Krásný & Gourse, ; Kriel et al, ). In B. subtilis , (p)ppGpp inhibits the activity of the GTP biosynthesis enzymes, Gmk, HprT and GuaB.…”

“…In B. subtilis , (p)ppGpp inhibits the activity of the GTP biosynthesis enzymes, Gmk, HprT and GuaB. In E. coli , Gmk is not inhibited by (p)ppGpp, but GuaB and HprT are inhibited, albeit at higher levels of (p)ppGpp than in B. subtilis (Anderson et al, ; Liu et al, ).…”