(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains

Mohammad Roghanian,Katleen Van Nerom,Hiraku Takada,Julien Caballero-Montes,Hedvig Tamman,Pavel Kudrin,Ariel Talavera,Ievgen Dzhygyr,Simon Ekström,Gemma C Atkinson,Abel Garcia-Pino,Vasili Hauryliuk

doi:10.1016/j.molcel.2021.07.026

Mohammad Roghanian, Katleen Van Nerom + Show 10 more

Open Access

https://doi.org/10.1016/j.molcel.2021.07.026

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Abstract

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function invivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E.coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

Highlights

The alarmone nucleotides guanosine pentaphosphate and tetraphosphate—collectively referred to as (p) ppGpp—are central regulators of bacterial metabolism and stress responses, playing an important role in antibiotic tolerance and virulence (Gaca et al, 2015; Hauryliuk et al, 2015; Liu et al, 2015)
We show that activation by the alarmone is crucial for induction of the synthetic activity of E. coli RelA by starved ribosomes—an effect that is readily masked in biochemical assays by (p)ppGpp produced in situ in the test tube
Comparison of the DHDX kinetics of RelA’s NTD fragment (RelANTD) supplemented with substrates, with that of RelANTD supplemented with pppGpp combined with GDP and APCPP, yielded the very same negative DHDX hot-spot located in the hinge region that we have identified through experiments with RelANTDpppGpp (Figure S1C)

Summary

Introduction

The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp)—collectively referred to as (p) ppGpp—are central regulators of bacterial metabolism and stress responses, playing an important role in antibiotic tolerance and virulence (Gaca et al, 2015; Hauryliuk et al, 2015; Liu et al, 2015). Cellular (p)ppGpp levels are controlled by enzymes belonging to the RelA/SpoT Homologue (RSH) family (Atkinson et al, 2011). Long RSHs are comprised of an N-terminal enzymatic half (N-terminal domain region, NTD) and C-terminal regulatory half (C-terminal domain region, CTD). The NTD contains two domains: the (p)ppGpp hydrolysis domain (HD; the enzymatically inactive HD of RelA is referred to as pseudo-HD) and the (p)ppGpp synthesis (SYNTH) domain. The majority of bacterial species contain a single long ribosomeassociated bifunctional RSH—Rel—that has both (p)ppGpp synthesis and hydrolysis activities (Atkinson et al, 2011). RelA is a ribosomal factor that lacks (p)ppGpp hydrolysis

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