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

DnaK is a prokaryotic Hsp70 chaperone, with numerous functions in helping to fold nascent polypeptides and more generally in proteostasis. It also restores native structures to heat-shocked proteins in an ATP-hydrolysis-dependent manner. The structures of DnaK complexes with nucleotides, co-chaperones and small peptides have already been resolved. However, there are no structures of DnaK complexes with larger, mostly folded substrates, such as firefly luciferase (Fluc, 61 kDa), which impedes the understanding of the mechanism through which DnaK refolds such large proteins. Here, we generated a model of a DnaK-firefly luciferase complex with Alphafold3, and examined its dynamics with all-atom molecular dynamics simulations. In this complex, Fluc is immobilized under the DnaK alpha-helical lid against the NBD, not the SBDβ, contrary to the data reported in the literature for model peptides. The DnaK lid is positioned strategically over Fluc’s helix 405-411, which we recently determined to be the first (and likely the only) helix melted in Fluc at 42 °C. We simulated the interaction between DnaK and the helix in its native and misfolded state and found that during the lid translocation toward the SBDβ, only the melted helix follows the lid and is actively pulled out from Fluc, while the native helix is not dislocated. These observations suggest a new model for the DnaK chaperone mechanism, where the alpha helical lid forms hydrogen bonds to the protein segment to be structurally tested. Lid pulls out only highly deformable misfolded helices, allowing them to refold into their native structures, and does not pull out those that are correctly folded because they are not deformable.