TY - JOUR

T1 - Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel

AU - Pausch, Patrick

AU - Abdelshahid, Maha

AU - Steinchen, Wieland

AU - Schäfer, Heinrich

AU - Gratani, Fabio Lino

AU - Freibert, Sven Andreas

AU - Wolz, Christiane

AU - Turgay, Kürşad

AU - Wilson, Daniel N.

AU - Bange, Gert

N1 - Funding information: G.B. D.N.W. and K.T. are grateful for financial support from the DFG priority program SPP1879. G.B. and P.P. acknowledge the excellent support by the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We thank Susanne Reider, Otto Berninghausen, and Roland Beckmann (University of Munich, Germany) for help with cryo-EM data collection. We acknowledge the Core Facility of Protein Spectroscopy and Protein Biochemistry of the Philipps-University Marburg. We thank Nils Mais for assistance during the revision of the manuscript. P.P. purified proteins, performed the pull-down assays and MALS experiments, and determined the crystal structure. M.A. performed the cryo-EM analysis. W.S. performed the biochemical activity assays. H.S. performed in vivo experiments. F.L.G. performed the bacterial 2-hybrid experiment. S.-A.F. and P.P. performed the Bio-layer Interferometry experiments. All authors contributed to the experimental design and analyzed the data. P.P. K.T. D.N.W. and G.B. wrote the manuscript. The authors declare that they have no competing interests. G.B., D.N.W., and K.T. are grateful for financial support from the DFG priority program SPP1879 . G.B. and P.P. acknowledge the excellent support by the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We thank Susanne Reider, Otto Berninghausen, and Roland Beckmann (University of Munich, Germany) for help with cryo-EM data collection. We acknowledge the Core Facility of Protein Spectroscopy and Protein Biochemistry of the Philipps-University Marburg. We thank Nils Mais for assistance during the revision of the manuscript.

PY - 2020/9/15

Y1 - 2020/9/15

N2 - The stringent response enables metabolic adaptation of bacteria under stress conditions and is governed by RelA/SpoT Homolog (RSH)-type enzymes. Long RSH-type enzymes encompass an N-terminal domain (NTD) harboring the second messenger nucleotide (p)ppGpp hydrolase and synthetase activity and a stress-perceiving and regulatory C-terminal domain (CTD). CTD-mediated binding of Rel to stalled ribosomes boosts (p)ppGpp synthesis. However, how the opposing activities of the NTD are controlled in the absence of stress was poorly understood. Here, we demonstrate on the RSH-type protein Rel that the critical regulative elements reside within the TGS (ThrRS, GTPase, and SpoT) subdomain of the CTD, which associates to and represses the synthetase to concomitantly allow for activation of the hydrolase. Furthermore, we show that Rel forms homodimers, which appear to control the interaction with deacylated-tRNA, but not the enzymatic activity of Rel. Collectively, our study provides a detailed molecular view into the mechanism of stringent response repression in the absence of stress.

AB - The stringent response enables metabolic adaptation of bacteria under stress conditions and is governed by RelA/SpoT Homolog (RSH)-type enzymes. Long RSH-type enzymes encompass an N-terminal domain (NTD) harboring the second messenger nucleotide (p)ppGpp hydrolase and synthetase activity and a stress-perceiving and regulatory C-terminal domain (CTD). CTD-mediated binding of Rel to stalled ribosomes boosts (p)ppGpp synthesis. However, how the opposing activities of the NTD are controlled in the absence of stress was poorly understood. Here, we demonstrate on the RSH-type protein Rel that the critical regulative elements reside within the TGS (ThrRS, GTPase, and SpoT) subdomain of the CTD, which associates to and represses the synthetase to concomitantly allow for activation of the hydrolase. Furthermore, we show that Rel forms homodimers, which appear to control the interaction with deacylated-tRNA, but not the enzymatic activity of Rel. Collectively, our study provides a detailed molecular view into the mechanism of stringent response repression in the absence of stress.

KW - (p)ppGpp

KW - alarmone

KW - cryo-EM

KW - enzymatic regulation

KW - homodimerization

KW - RSH-type enzyme

KW - stringent response

KW - X-ray crystallography

UR - http://www.scopus.com/inward/record.url?scp=85090739672&partnerID=8YFLogxK

U2 - 10.1016/j.celrep.2020.108157

DO - 10.1016/j.celrep.2020.108157

M3 - Article

C2 - 32937119

AN - SCOPUS:85090739672

VL - 32

JO - Cell reports

JF - Cell reports

SN - 2211-1247

IS - 11

M1 - 108157

ER -