Oral Presentation The 6th Prato Conference on Pore Forming Proteins 2025

Structural and functional insights into the Pseudomonas putida pore-forming toxin Tke5 (123802)

Maialen Zabala-Zearreta 1 , Carmen Velázquez 2 , Alejandro Arce-Rodríguez 3 , Jon Altuna-Alvarez 1 , Jessica Rojas-Palomino 4 , Andony Flores-Ceron 3 , Citlaly Cando 3 , Adrián Ruiz 3 , Amaia González-Magaña 1 , María Queralt-Martín 4 , Antonio Alcaraz 4 , Patricia Bernal 3 , David Albesa-Jové 1 2 5
  1. Fundación Biofisika Bizkaia, Bilbao, BIZKAIA, Spain
  2. Bioquímica y Biología Molecular, UPV-EHU, Leioa, BIZKAIA, Spain
  3. Microbiología, Universidad de Sevilla, Sevilla, Spain
  4. Physics, University Jaume I, Castellón, Spain
  5. Ikerbasque, Basque Foundation for Science, Bilbao, BIZKAIA, Spain

Pseudomonas putida KT2440 is a plant-beneficial rhizobacterium that encodes multiple Type VI secretion systems (T6SSs) to outcompete phytopathogens in the rhizosphere [1]. Among its antibacterial effectors, Tke5 has been recently identified as a potent Pore-Forming Toxin (PFT) that disrupts ion homeostasis without causing large membrane damage [2]. Tke5 belongs to the BTH_I2691 protein family and harbors an N-terminal MIX motif [3], previously shown to be required for secretion by the Type VI secretion system (T6SS). We will present the first biophysical and structural studies for this novel PFT. Our results provide the first molecular insight into the mechanism of action of the BTH_I2691 protein family. We will report the unpublished 2.77 Å cryo-EM structure of Tke5 in complex with its adaptor protein Tap3, revealing a novel protein fold that mediates MIX-dependent interactions. Tap proteins act as chaperones, promoting effector loading onto the T6SS machinery [4]. Our functional dissection shows that the α-helical region of Tke5 is sufficient to trigger membrane depolarisation, while its β-rich region likely contributes to target specificity. These findings suggest a general mechanism for MIX-dependent effector recognition and recruitment by T6SS-associated adaptors, contributing to our fundamental understanding of a widespread yet understudied PFT family.

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  2. [2] Carmen Velázquez, Alejandro Arce-Rodríguez, Jon Altuna-Alvarez, Jessica Rojas-Palomino, Andony Flores-Ceron, Citlaly Cando, Adrián Ruiz, Amaia González-Magaña, María Queralt-Martín, Antonio Alcaraz, David Albesa-Jové, Patricia Bernal Tke5 is a novel Pseudomonas putida toxin that depolarises membranes killing plant pathogens bioRxiv 2025.01.11.632536; doi: https://doi.org/10.1101/2025.01.11.632536
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