Rational Engineering of the Anthrax Toxin Nanopore Interface for Orthogonal Peptide Classification

Area of Science:

• Nanotechnology
• Biomaterials Science
• Molecular Engineering

Background:

• High-performance biointerfaces require precise nanoscale molecular recognition.
• Wild-type (WT) nanopore channels often lack specificity for chemically similar analytes.

Purpose of the Study:

• To engineer the anthrax toxin protective antigen (PA) nanopore for enhanced molecular recognition.
• To create a tunable biointerface with orthogonal selectivity by modifying the φ-clamp active site.

Main Methods:

• Rational engineering of the φ-clamp constriction in the PA nanopore (F427A mutation).
• Utilizing WT and F427A variants in a multistage machine learning ensemble (XGBoost).

Main Results:

• The F427A variant, despite impaired translocation, achieved ~93% accuracy in classifying peptides that confound WT pores.
• The XGBoost ensemble leveraged complementary selectivity (WT for aromatics, F427A for polar residues) achieving an F1-score >0.91.
• Demonstrated enhanced specificity of the biointerface for targeted analytical applications.

Conclusions:

• Rational engineering of nanopore active sites can create tunable biointerfaces with orthogonal selectivity.
• Attenuating native transport function enhances specificity for targeted analytical applications.
• Proposes a design principle for multiplexed biomaterials.