Structural and dynamical basis for the interaction of HSP70-EEVD with JDP Sis1

We employed NMR spectroscopy to investigate the structure and dynamics of the class B J domain protein (JDP) of S. cerevisiae (Sis1) complexed with an EEVD peptide of HSP70. It is widely recognized that the interactions between the EEVD motif and Sis1 play a crucial role in the chaperone activity. Notably, the deletion of the EEVD impairs the ability of Sis1 to bind with HSP70, while leaving the interaction between the class A JDP Ydj1 and HSP70 unaffected. Leveraging the advantages of NMR, which is particularly suitable for studying transient interactions, we provide compelling evidence that the EEVD motif transiently engages multiple sites on Sis1. Our findings revealed that EEVD binds to two distinct sites within the C-terminal domain I (CTDI) of Sis1. The interaction at these sites plays a crucial role in anchoring HSP70 to Sis1 at site I, as well as displacing the client protein at site II. Notably, site II is also the binding site for the client protein, and its displacement occurs through competition with the binding to site II. In addition to these interactions, we observed that EEVD, as a transient electrostatic binder, also interacts with the J domain and the GF-rich loop located between the J domain and -helix 6. We propose that the interaction between EEVD and Sis1 facilitates the dissociation of -helix 6, promoting a conformational state that is more favorable for interaction with HSP70 at the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) interface. We also employed -synuclein as a substrate to investigate the competitive nature between EEVD and the client protein. Our experimental findings provided evidence supporting the interaction of EEVD with the client protein at multiple sites. Our findings contribute essential insights into the mechanistic cycle of class B JDPs, paving the way toward a more complete understanding of the primary function of Sis1, which is the transfer of the client protein to HSP70, where multiple site transient interactions play a collective role.