Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK

TLDR

The 70‑kDa heat‑shock protein DnaK, like other Hsp70 family members, facilitates protein folding, interaction, and translocation by binding unfolded polypeptide segments through a substrate‑binding domain and an ATPase domain that mediates substrate exchange. The 2.0‑Å crystal structure of a peptide bound to DnaK’s substrate‑binding domain shows an extended peptide threaded through a loop‑defined channel in a β‑sandwich subdomain, with an α‑helical domain stabilizing the complex and rotating in a second lattice, supporting a latch‑like mechanism that prolongs substrate binding.

Abstract

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 Å resolution. The structure consists of a β-sandwich subdomain followed by α-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the β sandwich. An α-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.