The study reports the 2.0 Å resolution crystal structure of the substrate-binding unit of DnaK, a bacterial 70-kilodalton heat shock protein (hsp70) involved in protein folding and chaperone functions. The structure reveals a two-domain unit, with a β-sandwich subdomain followed by α-helical segments. The peptide is bound in an extended conformation through a channel defined by loops from the β-sandwich. An α-helical domain stabilizes the complex but does not directly contact the peptide. The structure shows conformational variations that suggest a latch mechanism for maintaining long-lived complexes. The substrate-binding unit of DnaK consists of two parts: a β-sandwich subdomain and an α-helical domain. The β-sandwich subdomain comprises residues 393 to 502 arranged in two sheets with four antiparallel β-strands in each. The α-helical domain is rotated in the molecules of a second crystal lattice, suggesting a conformation-dependent substrate binding mechanism. The structure reveals characteristics of the substrate-binding site, with exposed hydrophobic residues being generally required for substrate binding. The peptide binds to DnaK in an extended conformation, with interactions centered on Leu4, which is completely buried in a deep pocket. The binding interactions involve van der Waals contacts and hydrogen bonds, with the central site 0 being a crucial determinant of peptide binding. The structure also shows that the substrate peptide is bound in a hydrophobic pocket, with Leu4 occupying a pocket of ~5 Å wide and 3 Å deep. The study compares the structure of DnaK with other hsp70 proteins, showing high conservation in the β-subdomain and less conservation in the α-helical domain. The structure reveals that the substrate-binding channel is formed by loops L1,2 and L3,4, with the peptide bound in an extended conformation. The conformational variations between the two crystal lattices suggest a mechanism for substrate exchange, with the α-domain acting as a lid that must be displaced for binding and release. The study also discusses the functional implications of the structure, including the role of the ATPase domain and DnaJ in affecting the equilibrium among conformers. The structure provides insights into the conformational changes that occur during substrate binding and release, with the ATP-bound state having the α-helical lid domain closed and the ADP-bound state having it displaced. The findings suggest that the conformational changes are essential for the function of hsp70 proteins in protein folding and chaperone activities.The study reports the 2.0 Å resolution crystal structure of the substrate-binding unit of DnaK, a bacterial 70-kilodalton heat shock protein (hsp70) involved in protein folding and chaperone functions. The structure reveals a two-domain unit, with a β-sandwich subdomain followed by α-helical segments. The peptide is bound in an extended conformation through a channel defined by loops from the β-sandwich. An α-helical domain stabilizes the complex but does not directly contact the peptide. The structure shows conformational variations that suggest a latch mechanism for maintaining long-lived complexes. The substrate-binding unit of DnaK consists of two parts: a β-sandwich subdomain and an α-helical domain. The β-sandwich subdomain comprises residues 393 to 502 arranged in two sheets with four antiparallel β-strands in each. The α-helical domain is rotated in the molecules of a second crystal lattice, suggesting a conformation-dependent substrate binding mechanism. The structure reveals characteristics of the substrate-binding site, with exposed hydrophobic residues being generally required for substrate binding. The peptide binds to DnaK in an extended conformation, with interactions centered on Leu4, which is completely buried in a deep pocket. The binding interactions involve van der Waals contacts and hydrogen bonds, with the central site 0 being a crucial determinant of peptide binding. The structure also shows that the substrate peptide is bound in a hydrophobic pocket, with Leu4 occupying a pocket of ~5 Å wide and 3 Å deep. The study compares the structure of DnaK with other hsp70 proteins, showing high conservation in the β-subdomain and less conservation in the α-helical domain. The structure reveals that the substrate-binding channel is formed by loops L1,2 and L3,4, with the peptide bound in an extended conformation. The conformational variations between the two crystal lattices suggest a mechanism for substrate exchange, with the α-domain acting as a lid that must be displaced for binding and release. The study also discusses the functional implications of the structure, including the role of the ATPase domain and DnaJ in affecting the equilibrium among conformers. The structure provides insights into the conformational changes that occur during substrate binding and release, with the ATP-bound state having the α-helical lid domain closed and the ADP-bound state having it displaced. The findings suggest that the conformational changes are essential for the function of hsp70 proteins in protein folding and chaperone activities.