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

Van Nerom, Katleen ; Ainelo, Andres ; Coppieters 't Wallant, Kyo ; Talavera-Perez, Ariel ; Echemendia-Blanco, Dannele ; Peeters, Sarah ; El Khalfaoui Oulali, Brahim ; Tamman, Hedvig ; Kurata, Tatsuaki LU and Roghanian, Mohammad LU orcid , et al. (2026) In Nature Communications
Abstract

Long RSH enzymes, Rel and RelA, are master regulators of bacterial (p)ppGpp alarmones levels. Bifunctional Rel transitions between a compact hydrolysis-competent (HD
ON) state, a relaxed catalytically inactive (HD
OFF/SYNTH
OFF) state, and an elongated synthesis-competent (SYNTH
ON) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIA
Ntr, 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... (More)

Long RSH enzymes, Rel and RelA, are master regulators of bacterial (p)ppGpp alarmones levels. Bifunctional Rel transitions between a compact hydrolysis-competent (HD
ON) state, a relaxed catalytically inactive (HD
OFF/SYNTH
OFF) state, and an elongated synthesis-competent (SYNTH
ON) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIA
Ntr, 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 SYNTH
ON state, enhancing synthesis while suppressing hydrolysis, whereas Nb585 traps Rel in a hydrolysis-competent HD
ON/SYNTH
OFF 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.

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organization
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type
Contribution to journal
publication status
epub
subject
in
Nature Communications
publisher
Nature Publishing Group
external identifiers
  • pmid:42129208
  • scopus:105045202406
ISSN
2041-1723
DOI
10.1038/s41467-026-73059-3
language
English
LU publication?
yes
id
bfa6c700-fdfb-4af3-9dc3-a715e270cfbb
date added to LUP
2026-05-15 19:05:23
date last changed
2026-09-07 04:52:50
@article{bfa6c700-fdfb-4af3-9dc3-a715e270cfbb,
  abstract     = {{<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<br>
 ON) state, a relaxed catalytically inactive (HD<br>
 OFF/SYNTH<br>
 OFF) state, and an elongated synthesis-competent (SYNTH<br>
 ON) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIA<br>
 Ntr, 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 SYNTH<br>
 ON state, enhancing synthesis while suppressing hydrolysis, whereas Nb585 traps Rel in a hydrolysis-competent HD<br>
 ON/SYNTH<br>
 OFF 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.<br>
 </p>}},
  author       = {{Van Nerom, Katleen and Ainelo, Andres and Coppieters 't Wallant, Kyo and Talavera-Perez, Ariel and Echemendia-Blanco, Dannele and Peeters, Sarah and El Khalfaoui Oulali, Brahim and Tamman, Hedvig and Kurata, Tatsuaki and Roghanian, Mohammad and Martens, Chloé and Pardon, Els and Steyaert, Jan and Hauryliuk, Vasili and Garcia-Pino, Abel}},
  issn         = {{2041-1723}},
  language     = {{eng}},
  month        = {{05}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Nature Communications}},
  title        = {{Nanobody-mediated control of long RSH Rel and RelA catalysis by restriction of their conformational landscape}},
  url          = {{http://dx.doi.org/10.1038/s41467-026-73059-3}},
  doi          = {{10.1038/s41467-026-73059-3}},
  year         = {{2026}},
}