The structure of SpoT reveals evolutionary tuning of enzymatic output through constraint of the conformational landscape

Abstract

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyses (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 intra-molecular regulation of “long”-RSH 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 specialisation of long-RSHs towards either synthesis or hydrolysis: while the short and structured Core of SpoT stabilises the τ-state priming the HD domain for (p)ppGpp hydrolysis, the longer, more dynamic Core of RelA destabilises the τ-state precluding (p)ppGpp hydrolysis and priming RelA for synthesis