Topology and dynamics of the 10 kDa C‐terminal domain of DnaK in solution

Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N‐terminal ATPase domain, a 17 kDa peptide‐binding domain, and a 10 kDa C‐terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C‐terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C‐terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high‐resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C‐terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full‐length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze ¹⁵N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle‐like structure in solution.