Neutralization Mechanism of a HipA-like Toxin Targeting Isoleucyl-tRNA Synthetase

The HipA toxin from type II HipBA toxin-antitoxin (TA) system targets and inactivates specific cellular components to inhibit bacterial growth. While the molecular targets and neutralization mechanisms of several HipBA-like systems have been well characterized, their structural and functional diversity remains poorly understood. Here, we investigate a HipBA-like module from Pseudomonas fluorescens (HipBA_Pf), where the HipB_Pf antitoxin features a long, disordered C-terminal region in the absence of HipA_Pf. Using X-ray crystallography, AlphaFold modeling and mutagenesis assays, we show that upon binding to HipA_Pf, part of this C-terminal region forms two α-helices that are essential for both the interaction with and neutralization of the HipA_Pf toxin. Importantly, HipB_Pf binding blocks the ATP binding sites of HipA_Pf, potentially by inducing a conformational change in the HipA_Pf N1 subdomain via its C-terminal α6 helix. Finally, we also discovered that HipA_Pf (clade VI in the "Hip tree"), specifically phosphorylates isoleucyl-tRNA synthetase at Ser604, strongly inhibiting its aminoacylation activity. Collectively, our findings reveal the critical role of the HipB_Pf C-terminal region in toxin binding and neutralization, while also highlighting the evolutionarily divergent substrate preferences of HipA-like toxins.