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The dimeric protein Spy has a molecular weight of 32 kDa and is a good model for structurally understanding the mechanism of IDRs in chaperone action by NMR spectroscopy techniques. The ATP-independent chaperone Spy harbors long intrinsically disordered N and C termini, and their influence on Spy’s chaperone action remains enigmatic. However, high-molecular-weight proteins are a challenge to resolve by NMR and thus, it has been difficult to structurally elucidate the roles of IDRs in canonical chaperones, such as Hsp70 and GroEL. Nuclear magnetic resonance (NMR), on the other hand, has the unique advantage of providing detailed residue-by-residue information of protein disorder, making it highly suitable for investigating dynamic IDRs in chaperones 14. In addition, the high flexibility of IDRs hinders the study of IDRs by traditional biochemical and biophysical methods. A major challenge is that chaperone clients are prone to aggregation, which limits kinetic and structural analysis. Several “conditionally disordered” chaperones, such as Hsp26, Hsp33, and HdeA, rely on order-to-disorder conformational changes to bind and protect various clients from stress conditions 13.ĭespite increased research on the function of IDRs in chaperones, detailed kinetic and structural characterization of IDRs in chaperones is still lacking. The disordered C terminus of GroEL actively disrupts kinetically trapped, locally misfolded regions in the client proteins to facilitate their refolding 12. For example, the disordered C terminus of DnaK provides a binding motif with weak affinity for the clients, maintaining the ability of DnaK to support bacterial growth under thermal stress 11. Researchers have observed interactions between chaperone IDRs and client proteins and have noted the functional importance of IDRs in chaperone actions 10. The feature of molecular recognition presented by IDRs fits well with the promiscuous nature of molecular chaperones, which often need to recognize and bind to diverse kinds of proteins. IDRs exist in a heterogeneous ensemble of conformations and play various roles, including protein complex assembly, post-translational modification, signal transduction, substance transportation, and molecular recognition 7, 8, 9.
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It is estimated that 36.7% of the amino acid sequences in chaperones can be classified as “intrinsically disordered regions” (IDRs, i.e., protein regions lacking fixed tertiary structures) 6. To maintain proteostasis, molecular chaperone proteins, which are fundamental to protein quality control systems in cells, exert multiple functions including de novo folding of nascent polypeptides, preventing aggregation of folding intermediates, disassembling protein aggregates, and directing the degradation of off-pathway misfolded proteins 4, 5. Imbalance in protein homeostasis (“proteostasis”) can lead to protein misfolding and aggregation, which is associated with a variety of human diseases such as peripheral amyloidosis, type II diabetes, cancer, cardiovascular diseases, and many neurodegenerative disorders 1, 2, 3. Our results reveal the mechanism by which Spy releases clients independent of energy input, thus enriching the current knowledge on how ATP-independent chaperones release their clients and highlighting the importance of synergy between IDRs and structural domains in regulating protein function. This intramolecular interaction results in a dynamic competition of the N terminus with the client for binding to Spy, which promotes client discharge. With NMR spectroscopy and molecular dynamics simulations, we find that the N terminus can bind transiently to the client-binding cavity of Spy primarily through electrostatic interactions mediated by the N-terminal D26 residue. Here, we discover that the disordered N terminus of the prototype chaperone Spy facilitates client release.
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However, how IDRs impact chaperone action remains poorly understood. Molecular chaperones play a central role in regulating protein homeostasis, and their active forms often contain intrinsically disordered regions (IDRs).