Coppieters 't Wallant, K. | bioRxiv Science

bioRxiv2025

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

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 (HDON) state and an elongated, synthesis-competent (SYNTHON) state. The equilibrium between these states is modulated by factors such as "starved" ribosomes and regulatory proteins DarB, EIIANTR, 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. Nanobodies that lock the TGS domain of RelA and prevent its activation by deacylated tRNA on starved ribosomes, strongly inhibit (p)ppGpp synthesis and suppress the virulence of E. coli in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps Rel in a HDON 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 enzymes conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators.

microbiology↗

bioRxiv2024

A versatile bacterial innate immunity protein directly senses two disparate phage proteins

Eukaryotic innate immune systems use pattern recognition receptors (PRRs) to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages1-6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have only been found to sense a single trigger during infection, suggesting a one-to-one relationship between bacterial PRRs and their ligands7-11. Here, we demonstrate that the anti-phage defense protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the remarkable versatility of an immune sensory domain, which may be a common property of anti-phage defense systems and enable them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbor both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phage can only fully evade CapRelSJ46 defense when both triggers are mutated. Our work reveals how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defense systems and complex coevolutionary relationships between them and their phage-encoded triggers.

microbiology↗