Functional Dynamics of Hsp70/CHIP/E2-Ub Complex in Ubiquitination of Substrate Proteins.

Heat shock protein (Hsp) 70 is the main mediator of protein quality control in the cytosol of eukaryotic cells. One of the major functions of Hsp70 is mediate the ubiquitination of client proteins which subsequently leads to proteasomal degradation. This process occurs via a multiprotein complex composed of Hsp70, carboxy terminus of Hsc70 interacting protein (CHIP), ubiquitin-conjugating enzyme (E2), and ubiquitin (Ub). This complex is thought to be flexible, however, knowledge of the degree of dynamics are still lacking and detailed experimental evidence is needed. Here we present the models derived from Electron Paramagnetic Resonance (EPR) data and small-angle X-ray scattering (SAXS) ensembles for the complexes of Hsp70/CHIP/E2-Ub with ATP or ADP. The model of ATP bound Hsp70/CHIP/E2-Ub complex has an average distance of ~120 Å between the substrate-binding domain (SBD) and the E2-ubiquitin active site. The same distance of the multiprotein complex is found to be decreased to ~60 Å when Hsp70 is bound to ADP. Since the ADP bound conformation is considered as the active conformation that tightly binds client proteins, the alteration of the average distance between the Hsp70 SBD and the E2-Ub active site implies that the Lys residue of the substrate might need to be positioned within appropriate distance to span the gap in order to efficiently promote ubiquitination. Here, we used molecular dynamic simulations, double electron-electron resonance (DEER) EPR spectroscopy and in-vitroubiquitination assays with designed model substrate proteins that place lysine residues at variable positions. We found the dynamics of the Hsp70/CHIP/E2-Ubiquitin complex is playing a major role in the ubiquitination of various client proteins and these dynamics are necessary to overcome the distance barrier between E2-Ub active site and the Hsp70 SBD. Our data also suggests that the Hsp70/CHIP complex is capable of ubiquitinating a client protein substrate when lysine residues are placed at several disparate positions. In sum, our data reveals the functional dynamics of the Hsp70/CHIP/E2-Ub complex which enable ubiquitination of a client protein.