The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.
Significance
One of the key unresolved questions in the field of molecular chaperones is how they can actively unfold proteins. In this study, we discovered that the Hsp70/Hsp40 chaperone system completely unfolds a native soluble substrate protein, the ligand-binding domain of the glucocorticoid receptor, in a concerted action. Our high-resolution optical tweezers data show in real time how the substrate is attacked by the chaperone machinery. As soon as the hormone has left the binding pocket, up to five Hsp70/Hsp40 complexes bind and unfold the protein in a stepwise manner. This finding constitutes direct evidence that the chaperone machinery can bind to the folded core of the receptor, thus providing a mechanism for Hsp70-induced protein unfolding.
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