The structure of DarB in complex with Rel ^NTD reveals nonribosomal activation of Rel stringent factors

Abstract

Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP–binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB 2 :Rel NTD 2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.

Mechanisms of neutralization of toxSAS from toxin–antitoxin modules

“…Conversely, translation-targeting tRNA pyrophosphotransferases such as FaRel2 and CapRel are inhibited by pZFD interfering with the binding site for pyrophosphate donor ATP (Fig. 6b), which echoes the regulatory strategy used in the autoinhibition of multidomain 'long' (p)ppGpp-synthesizing housekeeping RSHs 12,27 . While the antitoxins and immunity proteins mediating the two neutralization strategies have diverged in sequence, the neutralizing elements display strong structural conservation.…”

(p)ppGpp – an important player during heat shock response

“…In the absence of c-di-AMP, DarB can interact with Rel and stimulate the synthesis of (p)ppGpp. These findings link the stringent response to c-di-AMP, another second messenger (Krüger et al 2021 , Heidemann et al 2022 , Ainelo et al 2023 ).…”

Role of (p)ppGpp in antibiotic resistance, tolerance, persistence and survival in Firmicutes

“…Rel can be allosterically activated by (p)ppGpp (Roghanian et al 2021 ), and its activity can be modulated by the second messenger c-di-AMP (Corrigan et al 2015 , Peterson et al 2020 , Krüger et al 2021 , Heidemann et al 2022 ). In some Firmicutes, Rel directly interacts with the c-di-AMP-binding protein DarB, thereby linking potassium deprivation with a stringent response (Krüger et al 2021 , Ainelo et al 2023 ). In Bacillus subtilis , lipid starvation (Pulschen et al 2017 ), heat shock (Schafer et al 2020 ), or certain antibiotics (Yang et al 2022 ) can also induce Rel-dependent (p)ppGpp synthesis by so far ill-defined mechanisms.…”