Using advanced NMR to reveal a new regulation mechanism of the Hsp70 chaperone

Protein chaperones are important players in our cells. The study of chaperones is a particularly challenging, as these typically operate through transient interactions on unstructured clients. NMR, however, is an ideal tool for this purpose, as it allows structural characterization even in the case of weak, short lived interactions involving disordered proteins. In our research we focused on Hsp70, a 70-kDa molecular chaperone that utilizes ATP hydrolysis to refold misfolded proteins and even to disaggregate amyloid fibrils. Its activity further depends on 80-90kDa co-chaperones - J-domain proteins (JDPs), which recruit Hsp70s to their substrates and stimulate ATP hydrolysis. There are two main classes of JDPs, which are structurally similar, yet have been shown to have different substrate specificities and cellular functions. The reasons behind this different specificity are largely unknown. We used NMR to answer these questions. Due to the large size of the proteins involved, beyond the limit of conventional NMR, we utilized methyl-TROSY NMR – the combination of selective ¹³CH₃ labelling of ILVM amino acids and TROSY-based pulse sequence. Using this approach, in combination with NMR NOEs and PRE experiments, we found that the 2 classes of JDPs interact differently not only with substrates, but also with Hsp70 itself. More specifically, class B JDPs displayed an intramolecular inhibition mechanism that does not appear in class A JDPs. This inhibition is lifted only upon JDP binding to the C-terminal region of Hsp70, enabling activation of this chaperone. Altogether, our results show that there is a regulation mechanism present only in class B JDPs which determines their interaction with Hsp70. This additional layer of regulation could help explain the need for class B to be present for efficient amyloid disaggregation.