Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding[S]

The oligomerization (and particularly dimerization) of Hsp70 proteins plays an important role in their chaperoning activities. Here, we report that human stress-inducible Hsp70 possesses the highest propensity among analyzed Hsp70 homologs to form dimers in the presence of ATP. ATP-bound Hsp70 assembles in solution as an antiparallel dimer closely resembling the dimeric structures captured in DnaK and BiP crystals. ATP-dependent Hsp70 dimerization is necessary for efficient Hsp40 interaction and is differentially affected by TPR cochaperone binding. [Display omitted] Highlights •Hsp70 homologs differ in their oligomeric properties in the presence of ATP.•Human inducible Hsp70 forms ATP-dependent anti-parallel dimers with high propensity.•Dimerization of ATP-bound Hsp70 is required for effective Hsp70-Hsp40 interaction.•ATP-dependent interaction with Tomm34 TPR cochaperone disrupts Hsp70 dimer. Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo. As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34