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

Abstract

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 0 position of GDP (or GTP) or remove the 3 0 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 Mn 2+ and mellitic acid. Rel Tt NTD crystallizes in space group P4 1 22, with unit-cell parameters a = b = 88.4, c = 182.7 Å , at 4 C and in space group P4 1 2 1 2, with unit-cell parameters a = b = 105.7, c = 241.4 Å , at 20 C. research communications Acta Cryst. (2019). F75, 561-569 Van Nerom et al. Rel stringent factor 565 research communications Acta Cryst. (2019). F75, 561-569 Van Nerom et al. Rel stringent factor 569

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA

“…Finally, our biochemical experiments using untagged full-length RelA fail to detect reduction in the ppGpp synthetic activity upon increase in enzyme’s concentration that is expected to drive the formation of inactive dimers (Figure 6). Our biochemical results are in good agreement with gel-filtration analysis of full-length Thermus thermophilus Rel that failed to detect protein dimerization (Van Nerom et al, 2019). Importantly, the said gel-filtration experiments was performed at unphysiologically high ionic strength that is necessary to keep relatively (in comparison to E. coli RelA) soluble T. thermophilus Rel in solution, and, therefore, should not be over-interpreted.…”

Dual role of a (p)ppGpp‐ and (p)ppApp‐degrading enzyme in biofilm formation and interbacterial antagonism

“…Pa SAH, MESH1 as well as Rel seq and Rel Tth harbor the amino acid residues critical for coordination of a divalent metal ion cofactor (i.e., HD motifs 2, 3, and the aspartate of HD5, Figure ) in similar structural arrangements. Likewise, Pa SAH exhibited a strong preference for Mn 2+ as a cofactor for (p)ppGpp hydrolysis over Mg 2+ in vitro, the same preference documented biochemically for C. glutamicum SAH (Ruwe et al., 2018), Dm MESH1 and Hs MESH1 (Sun et al., 2010) and the bifunctional Rel enzymes from S. equisimilis (Mechold et al., 2002), Mycobacterium tuberculosis (Avarbock et al., 2000), B. subtilis (Takada et al., 2020), T. thermophilus (Van Nerom et al., 2019), and Staphylococcus aureus (Yang et al., 2019). The concentration of Mn 2+ in vivo is estimated to be in the low micromolar range (Foster et al., 2014) and thus, 1,000‐fold less than the concentration necessary to promote hydrolytic activity in our in vitro assays (1–10 mM).…”

“…The concentration of Mn 2+ in vivo is estimated to be in the low micromolar range (Foster et al., 2014) and thus, 1,000‐fold less than the concentration necessary to promote hydrolytic activity in our in vitro assays (1–10 mM). Similar high concentrations of Mn 2+ were required for the in vitro activity of other (p)ppGpp hydrolases (see above) despite the dissociation constant of Rel Tth /Mn 2+ was quantified with 420 nM (Van Nerom et al., 2019). It seems possible that the activity of Pa SAH and other (p)ppGpp hydrolases observed in vitro would drastically overestimate their in vivo efficiency.…”

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

“…Next, we characterized the effects of substrates (GDP or GTP) as well as regulators (ribosomal complexes, tRNA, ppGpp and pppGpp) on the synthesis activity of Rel. Since Mn 2+ is universally essential for hydrolysis activity of long RSHs such as M. tuberculosis Rel ( 8 ), S. equisimilis Rel ( 16 ) T. thermophilus Rel ( 45 ) and E. coli SpoT ( 46 ), we characterized the synthesis activity of the full-length Rel in the absence of divalent manganese ions in order to avoid possible underestimation of the synthesis efficiency due to concomitant (p)ppGpp hydrolysis.…”