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Updated: Aug 18, 2025

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays
Published on: August 9, 2024
Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint
Hedvig Tamman1, Karin Ernits2,3,4, Mohammad Roghanian2,4,5
1Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB), Boulevard du Triomphe, Brussels, Belgium. hedvig.tamman@ulb.be.
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.
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