Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint

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 tau-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 tau-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the tau-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

Automated summary

Stringent factors regulate bacterial cell reprogramming by increasing the level of (p)ppGpp. This study presents the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and reveals the intramolecular regulation mechanism of 'long'-stringent factors. The Core subdomain plays a key role in determining the specialization of long RelA-SpoT homologs towards synthesis or hydrolysis.