Structural and functional insights into acidophilic helicases as DNA-unwinding motors.

In motor-assisted nanopore sensing, increasing the salt concentration improves the signal-to-noise ratio (SNR); however, helicases used as motor proteins generally fail to sustain efficient unwinding under high-salt conditions. In this study, we characterized the structure and function of AAA+ ATPase helicases from acidophiles. Bioinformatic analysis indicated that such helicases are broadly distributed among acidophilic bacteria. A representative helicase from IMPORTANCE: Nanopore sensing is a powerful approach for detecting and analyzing DNA at the single-molecule level, but its performance relies on specialized motor proteins that control DNA movement. A major challenge is that most available helicases lose activity in the high-salt conditions required to enhance signal quality. In this study, we characterized and engineered a helicase from an acidophilic bacterium that naturally thrives in extreme environments. By resolving its structure and stabilizing its flexible domains, we created a variant that remains functional under salt levels where conventional helicases fail, achieving a 6-fold increase in tolerance. These findings highlight extremophile enzymes as promising resources for designing robust molecular motors, expanding the toolbox for nanopore-based sensing and related biotechnological applications.