uptake plots RelCT-NTD with nanobodies 585 and 898

Long RSH enzymes, Rel and RelA, are the master regulators of (p)ppGpp alarmone levels in bacteria. Their catalytic activity is governed by transitions between a compact, hydrolysis-competent (HD^ON) state and an elongated, synthesis-competent (SYNTH^ON) state. The equilibrium between these states is modulated by factors such as “starved” ribosomes and regulatory proteins DarB, EIIA^NTR, ACP and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by trapping Rel/RelA enzymes in distinct conformational states. Nanobody Nb94 locks the TGS domain of E. coli RelA, preventing its activation by deacylated tRNA on starved ribosomes and thereby inhibiting (p)ppGpp synthesis. Nb898 stabilizes C. tepidum Rel in the open SYNTH^ON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps C. tepidum Rel in the HD^ON conformation, strongly inhibiting alarmone synthesis while promoting (p)ppGpp hydrolysis. Structural and biochemical analyses reveal that nanobodies, like natural allosteric regulators, act by restricting the RSH enzyme’s conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators.