The glucocorticoid receptor (GR) is an important transcription factor and drug target linked to a variety of biological functions and diseases. It is one of the most stringent physiological clients of the Hsp90/Hsp70/Hsp40 chaperone system. In this study, we used single-molecule force spectroscopy by optical tweezers to observe the interaction of the GR’s ligand-binding domain (GR-LBD) with the Hsp70/Hsp40 chaperone system (Hsp70/40). We show in real time that Hsp70/40 can unfold the complete GR-LBD in a stepwise manner. Each unfolding step involves binding of an Hsp70 to the GR-LBD and subsequent adenosine triphosphate (ATP) hydrolysis, stimulated by Hsp40. The kinetics of chaperone-mediated unfolding depend on chaperone concentrations as well as the presence of the nucleotide exchange factor BAG1. We find that Hsp70/40 can stabilize new unfolding intermediates, showing that Hsp70/40 can directly interact with the folded core of the protein when working as an unfoldase. Our results support an unfolding mechanism where Hsp70 can directly bind to folded protein structures and unfold them upon ATP hydrolysis. These results provide important insights into the regulation of GR by Hsp70/40.
Keywords: Hsp70; glucocorticoid receptor; optical tweezers; protein folding; single-molecule.