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.

Rel与RelA属于长链RSH酶（long RSH enzymes）家族，是调控细菌体内(p)ppGpp告警素水平的核心主控因子。其催化活性由两种构象状态间的动态转变所调控：一种是紧凑的、具备水解活性的（HD^ON）构象，另一种是延展的、具备合成活性的（SYNTH^ON）构象。这两种构象间的平衡可被多种因子调控，包括饥饿状态核糖体以及调控蛋白DarB、EIIA^NTR、ACP与YtfK。本研究中，我们鉴定并表征了一类骆驼科纳米抗体（camelid nanobodies），它们可通过将Rel/RelA酶捕获于特定构象状态，从而发挥选择性变构调控功能。纳米抗体Nb94可结合大肠杆菌（E. coli）RelA的TGS结构域，阻断饥饿状态核糖体上的去酰化tRNA对其的激活作用，进而抑制(p)ppGpp的合成。纳米抗体Nb898可将嗜热栖热菌（C. tepidum）Rel酶稳定于开放的SYNTH^ON构象，在增强其合成活性的同时抑制水解活性。与之相反，纳米抗体Nb585可将嗜热栖热菌（C. tepidum）Rel酶捕获于HD^ON构象，在强烈抑制告警素合成的同时促进(p)ppGpp的水解。结构与生化分析表明，与天然变构调控因子类似，纳米抗体通过限制RSH酶的构象空间发挥调控作用。本研究结果确立了纳米抗体作为解析RSH酶功能的强效工具，并为开发基于蛋白质的RSH调控剂提供了潜在研发线索。