33ZJ

The crystal structure of the polymorphic toxin PT9 and its cognate immunity PIM9 complex

Summary for 33ZJ

• Entry DOI: 10.2210/pdb33zj/pdb
• Descriptor: PIM9 from Cedecea neteri, PT9 from Cedecea neteri (3 entities in total)
• Functional Keywords: bacterial toxins, polymorphic toxins, antimicrobials, toxin
• Biological source: Cedecea neteri; More
• Total number of polymer chains: 2
• Total formula weight: 27579.05

Authors

Tzarum, N.,Weisz, J.,Fraenkel, R.,Cahana, I. (deposition date: 2026-09-01, release date: 2026-11-04)

Primary citation

Fraenkel, R.,Cahana, I.,Sivan, T.,Fisher, T.,Nadav, H.,Bruchim, S.,Weisz, J.,Deouell, N.,Cheskis, S.,Shalom, M.,Imbert, L.,Oppenheimer-Shaanan, Y.,Levy, A.,Tzarum, N.
Modular Platform for Recombinant Production of Highly Toxic Bacterial Proteins.
Microb Biotechnol, 19:e70453-e70453, 2026

PubMed Abstract: Bacterial protein toxins constitute a vast, largely untapped reservoir of potent bioactive molecules for antimicrobial discovery and biotechnology. However, their intrinsic toxicity often prevents recombinant expression in microbial hosts, limiting biochemical and functional characterization. Here, we present a modular platform for the recombinant production of highly toxic bacterial proteins based on covalent fusion of polymorphic toxins (PTs) to their cognate immunity proteins (PIMs), enabling transient toxin neutralization during expression. This strategy enabled recombinant production and purification of three previously refractory toxin families, PT7, PT8 and PT9. We further streamlined the workflow by introducing intracellular protease-mediated cleavage of the toxin-immunity fusion, reducing purification from four steps to two and increasing the recovered yield of PT9 from approximately 1-2 mg/L to ~4 mg/L. Extending the approach to PT37 further showed that computational redesign of the immunity protein and fusion architecture can enable production when the native fusion is insufficient. Purified PT7 and PT8 retained nuclease activity following denaturation and refolding, while refolded PT9 was structurally validated by X-ray crystallography, demonstrating recovery of functionally and structurally competent proteins. Together, these findings establish a modular strategy for recombinant production of highly toxic bacterial proteins and facilitate their biochemical and functional characterization.

PubMed: 42888866
DOI: 10.1111/1751-7915.70453

Experimental method

X-RAY DIFFRACTION (1.54 Å)