Interdomain communication suppressing high intrinsic ATPase activity of Sse1 is essential for its co-disaggregase activity with Ssa1.

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70-like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single-molecule FRET, small angle X-ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70-like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open-lid substrate-binding domain (SBD) in close contact with its nucleotide-binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP-hydrolysis-independent exhaustive interaction between two domains of Hsp110s, NBD-SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1-NBD compared to wild-type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1-mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co-chaperoning activity in disaggregase machinery, and stability of the protein.

Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

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 intradomain 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 nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone 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.