Abstact

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 alpha-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 alpha6 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.