The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis

The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3′ position of GDP (or GTP) or remove the 3′ pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (Rel_Tt^NTD) in the resting state and bound to nucleotides are described. Rel_Tt and Rel_Tt^NTD are monomers in solution that are stabilized by the binding of Mn²⁺ and mellitic acid. Rel_Tt^NTD crystallizes in space group P4₁22, with unit-cell parameters a = b = 88.4, c = 182.7 Å, at 4°C and in space group P4₁2₁2, with unit-cell parameters a = b = 105.7, c = 241.4 Å, at 20°C.