Presentation Information
[P01-107]Construction and characterization of a β-barrel nanopore derived from the transmembrane domain of a trimeric autotransporter adhesin
○Jun Sasahara1, Shogo Yoshimoto1, Zugui Peng2, Taehyun Hwang1, Iori Kobayashi1, Ryuji Kawano2, Katsutoshi Hori1 (1. Nagoya University (Japan), 2. Tokyo University of Agriculture and Technology (Japan))
Keywords:
Nanopore,Molecular dynamics simulation,Trimeric autotransporter adhesin,Type Vc secretion system
[Background]
Biological nanopores are powerful tools for single-molecule sensing, with applications in DNA sequencing and protein analysis. While secretion systems from Type Va and Type Vb classes have been successfully adapted as nanopores, the potential of Type Vc secretion systems, particularly trimeric autotransporter adhesins (TAAs), remains largely unexplored. TAAs such as AtaA from Acinetobacter sp. Tol 5 form a distinctive trimeric 12-stranded transmembrane β-barrel. In the native structure, the central lumen is occupied by a coiled-coil passenger domain during secretion. We hypothesized that removal of this lumen-occupying segment would generate a stable open-pore scaffold suitable for nanopore applications.
[Methods]
We engineered an open β-barrel nanopore, termed AtaApore, by genetically deleting the internal coiled-coil region of the AtaA transmembrane domain. Because heterologous expression in Escherichia coli was inefficient, the protein was synthesized using a cell-free expression system (PUREfrex 2.0). Pore formation was first evaluated using a liposomal permeation assay based on calcein encapsulation. Electrophysiological characterization was then performed using planar lipid bilayer recordings. In parallel, all-atom molecular dynamics simulations were conducted to evaluate structural stability (1,000 ns) and ion transport behavior (500 ns) under an applied potential of +150 mV.
[Results]
SDS–PAGE and Blue Native PAGE confirmed successful cell-free synthesis of AtaApore and its assembly into a trimeric complex. In the liposomal permeation assay, AtaApore induced substantial dye release, demonstrating membrane pore formation. Electrophysiological recordings revealed stable single-channel activity with a median conductance of 0.17 nS in 1 M KCl. This conductance is lower than that of established nanopores such as α-hemolysin, indicating a relatively narrow lumen that may be advantageous for sensing small analytes. Molecular dynamics simulations confirmed structural stability of the β-barrel and identified a defined constriction region formed by residues R3597 and R3622. These positively charged residues transiently interacted with chloride ions, modulating ion permeation through the pore.
[Conclusion]
This study demonstrates the successful construction and functional characterization of a nanopore derived from a Type Vc secretion system. AtaApore represents a new scaffold for nanopore engineering, characterized by a narrow lumen and a well-defined electrostatic constriction site. In addition to potential applications in biosensing, this engineered open β-barrel provides a simplified structural model for investigating transport mechanisms associated with trimeric autotransporter adhesins.
[Reference]
J. Sasahara, S. Yoshimoto, Z. Peng, T. Hwang, I. Kobayashi, R. Kawano, K. Hori. Construction and characterization of a nanopore derived from the transmembrane domain of a trimeric autotransporter adhesin. Frontiers in Bioengineering and Biotechnology 14, 1764864 (2026).
Biological nanopores are powerful tools for single-molecule sensing, with applications in DNA sequencing and protein analysis. While secretion systems from Type Va and Type Vb classes have been successfully adapted as nanopores, the potential of Type Vc secretion systems, particularly trimeric autotransporter adhesins (TAAs), remains largely unexplored. TAAs such as AtaA from Acinetobacter sp. Tol 5 form a distinctive trimeric 12-stranded transmembrane β-barrel. In the native structure, the central lumen is occupied by a coiled-coil passenger domain during secretion. We hypothesized that removal of this lumen-occupying segment would generate a stable open-pore scaffold suitable for nanopore applications.
[Methods]
We engineered an open β-barrel nanopore, termed AtaApore, by genetically deleting the internal coiled-coil region of the AtaA transmembrane domain. Because heterologous expression in Escherichia coli was inefficient, the protein was synthesized using a cell-free expression system (PUREfrex 2.0). Pore formation was first evaluated using a liposomal permeation assay based on calcein encapsulation. Electrophysiological characterization was then performed using planar lipid bilayer recordings. In parallel, all-atom molecular dynamics simulations were conducted to evaluate structural stability (1,000 ns) and ion transport behavior (500 ns) under an applied potential of +150 mV.
[Results]
SDS–PAGE and Blue Native PAGE confirmed successful cell-free synthesis of AtaApore and its assembly into a trimeric complex. In the liposomal permeation assay, AtaApore induced substantial dye release, demonstrating membrane pore formation. Electrophysiological recordings revealed stable single-channel activity with a median conductance of 0.17 nS in 1 M KCl. This conductance is lower than that of established nanopores such as α-hemolysin, indicating a relatively narrow lumen that may be advantageous for sensing small analytes. Molecular dynamics simulations confirmed structural stability of the β-barrel and identified a defined constriction region formed by residues R3597 and R3622. These positively charged residues transiently interacted with chloride ions, modulating ion permeation through the pore.
[Conclusion]
This study demonstrates the successful construction and functional characterization of a nanopore derived from a Type Vc secretion system. AtaApore represents a new scaffold for nanopore engineering, characterized by a narrow lumen and a well-defined electrostatic constriction site. In addition to potential applications in biosensing, this engineered open β-barrel provides a simplified structural model for investigating transport mechanisms associated with trimeric autotransporter adhesins.
[Reference]
J. Sasahara, S. Yoshimoto, Z. Peng, T. Hwang, I. Kobayashi, R. Kawano, K. Hori. Construction and characterization of a nanopore derived from the transmembrane domain of a trimeric autotransporter adhesin. Frontiers in Bioengineering and Biotechnology 14, 1764864 (2026).
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