Molecular mechanism of the Hsp70 Chaperone network

• Applicant: Professor Dr. Matthias Peter Mayer
• Subject Area: Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 462625623

The ATP-dependent 70 kDa heat shock proteins (Hsp70s) are the central hub of the cellular protein quality surveillance system. They are involved in a wide variety of protein folding processes. The versatility of Hsp70 is based on transient binding of their substrate binding domain (SBD) to short degenerative sequence motifs found in practically all proteins, on allosteric regulation of substrate binding by ATP-binding and hydrolysis in their nucleotide binding domain (NBD), on the assistance by J-domain proteins (JDPs) that target Hsp70s to their protein substrates and nucleotide exchange factors (NEFs) that regulate the lifetime of Hsp70-substrate complexes, and on cooperation with other chaperones of the small heat shock protein, the Hsp90 and Hsp100 families. The key questions that we ask in this project are: Why do Hsp70s have a tendency to oligomerize in the ADP bound state and how is the structure of this oligomeric complex? Why is a subset of Hsp70s dimeric in the ATP bound state? Why are many general JDPs dimeric proteins? Why are there at least five bona fide Hsp70s in the cytosol of human cells and how do the 30 JDPs in the cytosol of human cells target the right Hsp70 to the correct target? In the last funding period, we demonstrated that human Hsc70 oligomerizes by binding of the SBD of one Hsc70 to the interdomain linker of a second Hsc70 like to a substrate and we identified Hsc70 variants with different oligomerization propensity. Importantly, Hsc70 variants with higher oligomerization propensity had higher chaperone activity, suggesting that oligomerization is not a storage form but functionally relevant. We now want to get biochemical evidence that Hsc70 oligomerizes on substrates to substantiate the hypothesis that the purpose of oligomerization is the increase in entropic pulling force. Using our newly developed interaction assay, we mapped the interaction network of all human JDPs with all bona fide human Hsp70s. We now want to verify the sequence requirements for the JDP specificity that are deduced from our data. Using monomeric and heterodimeric variants of the E. coli JDP DnaJ we demonstrated that dimerization is beneficial but not absolutely required for E. coli growth at elevated temperatures and swimming and for refolding a denatured model substrate in vitro. We now want to evaluate the interaction of DnaJ with specific oligomeric substrates, to see whether they require the dimeric state.

• DFG Programme: Research Grants