Structural insights into substrate binding by the molecular chaperone DnaK

How substrate affinity is modulated by nucleotide binding remains a fundame ntal, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability t o support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding su bstrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta 3 are in significantly different conformations than observed in previo us structures of the bound DnaK substrate binding domain, leading to occlus ion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Take n together, our results suggest that conformational changes in the beta-dom ain itself contribute to the mechanism by which nucleotide binding modulate s substrate binding affinity.