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 Leptospirillum spp., designated LfDda, adopts a dimeric architecture at 3.5 Å resolution with a flexible tower domain while retaining conserved 1A and 2A domains, akin to well-studied motor (T4 Dda helicase). Biochemical assays demonstrated that the dimeric form of LfDda is functional, acting as a 5′–3′ directional helicase that can utilize various divalent metal ions as cofactors, with Mn2+ supporting the highest catalytic activity. Crosslinking of the flexible 1B and 2B domains yielded a monomeric variant capable of efficient DNA translocation at 600 mM KCl, representing a 6-fold enhancement in ionic tolerance. These findings suggest that helicases derived from extremophilic bacteria can be engineered to modulate DNA translocation behavior, providing a potential avenue for developing new motor proteins for nanopore and other DNA-unwinding applications.