Unveiling Acidophilic Secretin Nanopores for Ion Exclusion and Single‐Molecule Sensing

Abstract Biological nanopores formed by hepta‐ to nonameric assemblies create sub‐2 nm conduits that have revolutionized single‐molecule analytics, yet they remain constrained in resolution and analyte scope. Enlarged β‐barrel assemblies might serve as a promising solution, but no high‐order pore with both atomic definition and reliable membrane insertion has been available. Here, it is shown that the secretin AcGspD from the acidophile Acidithiobacillus caldus constitutes a 14‐mer nanopore scaffold with exceptional membrane‐insertion capacity. A 2.4 Å cryo‐electron microscopy (cryo‐EM) structure reveals a flat‐tipped constriction topped by interlocking lip‐ and tongue‐like loops and stabilized by a degenerate N‐terminal domain. An extended, aromatic‐rich membrane belt drives rapid, orientation‐correct insertion into lipid bilayers and enables robust reconstitution in droplet‐interface systems. Electrophysiological recordings demonstrate that wild‐type AcGspD is strongly cation‐selective yet effectively excludes Ca 2+ and Fe 3+ ions via hydrophobic gating at its central constriction. Substituting gate residues with polar, uncharged amino acids converts AcGspD into a single‐molecule sensor that docks homopolynucleotides and peptides and threads DNA in discrete, stepwise current signatures distinct from existing nanopores. These results establish secretins as an untapped class of programmable nanopores and position AcGspD as a durable platform for high‐resolution molecular analytics and size‐selective membrane technologies.