Indexed on: 02 Jun '16Published on: 02 Jun '16Published in: FEBS Journal
DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide binding domain (NBD) and the C-terminal substrate or peptide binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single molecule-Förster Resonance Energy Transfer (sm-FRET) measurements to capture distinct intra-domain conformational states of a region within the DnaK SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a non-hydrolysable ATP analogue, AMP-PNP imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The co-chaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the co-chaperone accelerates the substrate binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK SBD, leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures. This article is protected by copyright. All rights reserved.