DnaK refolds denatured proteins by actively pulling out their misfolded structural elements

ABSTRACT DnaK, a prokaryotic Hsp70 chaperone, plays a central role in proteostasis by restoring native structures to heat-denatured proteins in an ATP-hydrolysis–dependent manner. While structures of DnaK in complex with nucleotides, co-chaperones, and short peptides have been resolved, structures with larger, stably folded substrates—such as firefly luciferase (Fluc, 61 kDa)—are lacking, limiting mechanistic understanding of how DnaK refolds such proteins. Here, we generated models of the DnaK–Fluc complex using AlphaFold3 and evaluated their mechanistic relevance. In one of three major model clusters, Fluc is unexpectedly immobilized beneath the DnaK α-helical lid against the nucleotide-binding domain (NBD), rather than interacting primarily with the substrate-binding domain β (SBDβ), as commonly assumed. All-atom molecular dynamics simulations indicate that, in this configuration, the lid can engage a thermally destabilized Fluc helix (residues 405–411), which we recently identified as the first—and likely the only—helix to irreversibly melt at 42 °C. Upon binding, the lid forms extensive hydrogen-bonding interactions with the melted helix. These interactions persist during lid movement toward SBDβ (following ATP hydrolysis), enabling the lid to actively extract the helix from the Fluc surface. In contrast, simulations with the helix in its native folded state show that the lid cannot extract it, leaving the native structure unaffected. Equilibrium simulations further indicate that, once extracted and mechanically stretched, the melted helix can refold to its native conformation. Together, these findings suggest a revised mechanism for DnaK-mediated protein refolding, in which the α-helical lid selectively recognizes structurally compromised segments, forms stabilizing hydrogen bonds, and—powered by ATP hydrolysis—mechanically pulls them away from the protein surface to facilitate their refolding. SIGNIFICANCE DnaK is a model chaperone, which can reactivate thermally denatured proteins. Over the span of 40 years, significant findings have been made about DnaK’s structure, dynamics and interactions with its co-chaperones, the exact molecular mechanism by which DnaK refolds misfolded proteins remains a mystery. This work exploited Alphafold3 to generate atomistic models of complexes between DnaK and Firefly luciferase. Molecular dynamics simulations directly captured how DnaK may assist thermally denatured proteins by mechanically pulling out their misfolded helices. This study provides a new insight into the DnaK mechanism.