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

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 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.