Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint

Abstract

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of ‘long’-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

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

“…6B). The relaxed state (10) would be able to accommodate the HD domain moving away from SYNTH, resulting from the interaction with DarB. This is consistent with the significant drop in hydrolysis by Rel Bs upon binding to DarB.…”

“…Dedicated (p)ppGpp synthetases such as RelA, which are mainly under ribosome control and unlikely to interact with DarB homologs, have a very different conservation pattern in the β3 region that connects directly with DarB α1 (fig. S2B), which overlaps with a region of SYNTH involved in G nucleotide substrate specificity ( 38 ) and the N-cap of α13, which interacts with the RRM domain of Rel in the HD ON τ state (but not in RelA) ( 10 ). Thus, it appears that DarB has evolved to recognize a conserved multifunctional Rel hotspot rather than the interfaces having coevolved as such.…”

“…Off the ribosome, HD-competent RSHs SpoT and Rel assume a compact state that suppresses the SYNTH activity of the NTD through cis-autoinhibition mechanism (10,12). In this state, the CTD domains TGS and helical directly stimulate hydrolysis by the HD domain, while ZFD and RRM suppress the SYNTH activity by precluding the guanosine diphosphate (GDP) and adenosine triphosphate (ATP) substrate binding (10,12). Upon association with starved ribosomes-i.e., stalled ribosomal elongation complexes harboring a cognate deacylated tRNA in the A site-Rel/RelA assumes a highly elongated SYNTH-active state in which the CTD is decoupled from the NTD (18,(21)(22)(23).…”

“…The stringent factor RelA is a monofunctional ribosome-associated (p)ppGpp synthetase activated during amino acid starvation ( 7 ). SpoT is a potent (p)ppGpp hydrolase with a weak synthesis activity ( 8 , 9 ) that became monofunctional in the Moraxellaceae lineage ( 4 , 10 ). While the phylogenetic distribution of RelA and SpoT is limited to Beta- and Gammaproteobacteria ( 4 , 5 ), the most taxonomically widespread long RSH representative, Rel, is a bifunctional ribosome-associated (p)ppGpp synthetase/hydrolase that, similarly to RelA, senses amino acid starvation on the ribosome ( 11 , 12 ).…”

“…The regulatory C-terminal domain region (CTD) consists of four nonenzymatic domains: TGS, helical, ZFD, and RRM ( 4 , 18 ). Through extensive intramolecular contacts with the NTD via a core domain, the CTD controls the global conformational state of the enzyme to regulate its enzymatic output ( 10 , 12 , 13 , 19 , 20 ). Off the ribosome, HD-competent RSHs SpoT and Rel assume a compact state that suppresses the SYNTH activity of the NTD through cis-autoinhibition mechanism ( 10 , 12 ).…”