Functional Regulation of Hsp70 Allostery by Post‐Translational Modifications (PTMs)

The 70‐kDa heat shock proteins (Hsp70s) perform critical functions in the protein homeostasis network, such as assisting nascent chain folding, preventing protein misfolding and aggregation, facilitating assembly of complexes, and driving protein translocation. The function of Hsp70s relies on a conserved allosteric mechanism involving substrate binding and release modulated by ATP binding and hydrolysis, with the participation of co‐chaperones, including J‐proteins and nucleotide exchange factors. Many studies in recent years have demonstrated that post‐translational modifications (PTMs) play important roles in functional regulations of Hsp70s. As an example, a recent report showed that phosphorylation of Hsp70 T495 upon infection by Legionella pneumophila inhibits host protein translation. While the modulation of Hsp70 functions can be achieved by a variety of means, including tuning its allosteric landscape, altering its substrate binding properties, varying its co‐chaperone interaction, etc., the mechanism of functional regulation of Hsp70s through PTMs is still unknown. In the current study, by using nuclear magnetic resonance spectroscopy (NMR) and biophysical/biochemical assays based on phosphorylation‐mimicking mutant, we are able to elucidate the origin of functional regulation by T495 phosphorylation in Hsp70. Phosphorylation of T495 shifts Hsp70 to the conformation favored by ATP binding in which the nucleotide‐binding domain (NBD) and substrate‐binding domain (SBD) are intimately docked, thus reducing the substrate binding activity of the molecular chaperone. These results underline the regulation of Hsp70 function by modulating its allosteric landscape through PTMs. Support or Funding Information Supported by NIH grant GM118161