The article presents the structural analysis of substrate binding by the molecular chaperone DnaK, a member of the 70-kilodalton heat-shock protein (hsp70) family. DnaK and other hsp70 proteins promote protein folding, interaction, and translocation by binding to unfolded polypeptide segments. The crystal structure of a peptide complex with the substrate-binding unit of DnaK at 2.0 Å resolution reveals a β-sandwich subdomain and an α-helical domain. The peptide is bound in an extended conformation through a channel defined by loops from the β-sandwich, with the α-helical domain stabilizing the complex but not directly contacting the peptide. The α-helical domain is rotated in the molecules of a second crystal lattice, suggesting a model of conformation-dependent substrate binding with a latch mechanism for maintaining long-lived complexes. The structure provides insights into the function of hsp70 proteins, including their ATPase and substrate-binding activities, and their interactions with accessory chaperones like DnaJ and GrpE. The study also discusses the conservation and sequence differences among hsp70 proteins and the implications for substrate binding and release mechanisms.The article presents the structural analysis of substrate binding by the molecular chaperone DnaK, a member of the 70-kilodalton heat-shock protein (hsp70) family. DnaK and other hsp70 proteins promote protein folding, interaction, and translocation by binding to unfolded polypeptide segments. The crystal structure of a peptide complex with the substrate-binding unit of DnaK at 2.0 Å resolution reveals a β-sandwich subdomain and an α-helical domain. The peptide is bound in an extended conformation through a channel defined by loops from the β-sandwich, with the α-helical domain stabilizing the complex but not directly contacting the peptide. The α-helical domain is rotated in the molecules of a second crystal lattice, suggesting a model of conformation-dependent substrate binding with a latch mechanism for maintaining long-lived complexes. The structure provides insights into the function of hsp70 proteins, including their ATPase and substrate-binding activities, and their interactions with accessory chaperones like DnaJ and GrpE. The study also discusses the conservation and sequence differences among hsp70 proteins and the implications for substrate binding and release mechanisms.