Secreted nuclease effector neutralization by active site mimicry in Bacillota

Polymorphic toxins mediate interbacterial antagonism among competitors in the gut microbiome. Nuclease effectors, distantly related to the type VI-secreted Bacteroidales Tde, are enriched in human gut Bacillota. Tde mediates antagonism among Bacillota, and expression of the cognate immunity, Tdi, in recipients is protective. Crystal structures of Tde/Tdi complexes from two Bacillus spp. and Enterococcus quebecensis highlight a conserved mechanism of immunity. Tdi engages Tde with high-affinity, specific binding at an interface that features predominantly polar amino acids. A separate Tdi interface has a very highly conserved P(Φ) ₄ GG motif that structurally mimics and displaces a short helix in Tde’s active site, which contains the critical catalytic residues. An isolated P(Φ) ₄ GG motif peptide is sufficient for Tde nuclease activity inhibition at high concentrations. However, key residues at both the polar interface and P(Φ) ₄ GG are required for complete inhibition of nuclease activity and protection against toxicity. We propose a multivalent Tde/Tdi neutralization mechanism where an initial high-affinity interface increases the local concentration of Tdi’s P(Φ) ₄ GG motif, enabling it to displace the Tde active site through structural mimicry. The resulting conformational rearrangement of Tde increases its flexibility in solution and susceptibility to proteolysis, which may aid in eliminating the toxic effector.