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<p>Long RSH enzymes, Rel and RelA, are master regulators of bacterial (p)ppGpp alarmones levels. Bifunctional Rel transitions between a compact hydrolysis-competent (HD<sup>ON</sup>) state, a relaxed catalytically inactive (HD<sup>OFF</sup>/SYNTH<sup>OFF</sup>) state, and an elongated synthesis-competent (SYNTH<sup>ON</sup>) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIA<sup>Ntr</sup>, ACP, NirD and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by stabilizing Rel and RelA in defined conformational states. Nanobodies that sequester the TGS domain of RelA prevent activation by deacylated tRNA on starved ribosomes, strongly inhibiting (p)ppGpp synthesis and suppressing <i>Escherichia coli</i> virulence in an animal model. Nb898 stabilizes Rel in the open SYNTH<sup>ON</sup> state, enhancing synthesis while suppressing hydrolysis, whereas Nb585 traps Rel in a hydrolysis-competent HD<sup>ON</sup>/SYNTH<sup>OFF</sup> conformation. Structural and biochemical analyses show that nanobodies, like endogenous allosteric regulators, restrict the conformational landscape of long RSH enzymes, establishing them as powerful tools for dissecting RSH function and as frameworks for developing protein-based RSH modulators.</p>

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Nanobody-mediated control of long RSH Rel and RelA catalysis by restriction of their conformational landscape

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月13日

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  • Katleen Van Nerom,
  • Andres Ainelo,
  • Kyo Coppieters ‘t Wallant,
  • Ariel Talavera-Perez,
  • Dannele Echemendia-Blanco,
  • Sarah Peeters,
  • Brahim El Khalfaoui Oulali,
  • Hedvig Tamman,
  • Tatsuaki Kurata,
  • Mohammad Roghanian,
  • Chloé Martens,
  • Els Pardon,
  • Jan Steyaert,
  • Vasili Hauryliuk,
  • Abel Garcia-Pino

摘要

Long RSH enzymes, Rel and RelA, are master regulators of bacterial (p)ppGpp alarmones levels. Bifunctional Rel transitions between a compact hydrolysis-competent (HDON) state, a relaxed catalytically inactive (HDOFF/SYNTHOFF) state, and an elongated synthesis-competent (SYNTHON) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIANtr, ACP, NirD and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by stabilizing Rel and RelA in defined conformational states. Nanobodies that sequester the TGS domain of RelA prevent activation by deacylated tRNA on starved ribosomes, strongly inhibiting (p)ppGpp synthesis and suppressing Escherichia coli virulence in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis while suppressing hydrolysis, whereas Nb585 traps Rel in a hydrolysis-competent HDON/SYNTHOFF conformation. Structural and biochemical analyses show that nanobodies, like endogenous allosteric regulators, restrict the conformational landscape of long RSH enzymes, establishing them as powerful tools for dissecting RSH function and as frameworks for developing protein-based RSH modulators.