A fluorescent multi-domain protein reveals the unfolding mechanism of Hsp70

The proper folding of proteins is crucial for cellular function. Nascent polypeptide chains spontaneously fold into secondary and tertiary structures. Once released, proteins either function directly, translocate, or assemble into functional oligomers. Maintaining protein stability is essential in the crowded cellular environment. However, stress can lead to partial unfolding, exposing hydrophobic residues and causing misfolding and aggregation. These aggregates, varying in size, compactness, and solubility, often do not spontaneously refold after stress. Misfolding and aggregation are inherent to protein physics and have cytotoxic effects, particularly in neurons. Molecular chaperones like Hsp70s, Hsp60s, Hsp90s, and Hsp100s, aided by co-chaperones, use ATP hydrolysis to prevent and reverse protein aggregation. The complexity of this chaperone network has increased with proteome complexity throughout evolution. Significant evidence suggests that Hsp70s, Hsp100s, and Hsp60s act as polypeptide-unfolding enzymes, targeting stable misfolded and aggregated proteins. They use ATP to convert these substrates into unstable, unfolded monomers, which can then spontaneously refold. Hsp70 is a central coordinator in this protein repair machinery. Its activity depends on co-chaperones called J-domain proteins (JDPs), which accelerate the ATPase cycle, leading to strong Hsp70 binding and subsequent substrate unfolding. However, characterizing aggregated protein substrates is challenging due to the heterogeneity of non-native protein ensembles.

The literature extensively documents the roles of molecular chaperones in preventing protein aggregation and assisting in protein folding. Studies have shown that Hsp70, in conjunction with co-chaperones like Hsp40 and nucleotide exchange factors, plays a critical role in maintaining proteostasis. The ATPase cycle of Hsp70 is central to its function, cycling between high and low affinity states for substrates. The role of JDPs in targeting Hsp70 to misfolded proteins has also been established. However, understanding the exact mechanism of Hsp70 action on aggregated substrates has been hampered by the difficulty of characterizing these heterogeneous populations of misfolded proteins. Previous studies have often relied on indirect methods or used simplified systems, making it difficult to fully elucidate the details of the Hsp70 mechanism.

To overcome the challenges of studying heterogeneous protein aggregates, the researchers designed a novel reporter protein, MLucV. MLucV is a monomeric 120-kDa protein composed of a heat-labile and urea-sensitive mutant firefly luciferase core flanked by urea- and thermo-resistant fluorescent domains (mTFP1 and Venus). This design allows for the monitoring of protein conformation using Förster resonance energy transfer (FRET) and luciferase enzymatic activity. The researchers used urea and heat treatments to create reproducible, small, soluble MLucV aggregates. The action of the bacterial Hsp70 system (DnaK-DnaJ-GrpE) was then studied in detail, revealing the steps involved in disaggregating stable protein aggregates without the need for ClpB. Various techniques were employed, including FRET spectroscopy to monitor conformational changes, luciferase activity assays to assess protein functionality, negative stain electron microscopy to visualize aggregate morphology and size, size-exclusion chromatography-right-angle light scattering (SEC-RALS) to determine aggregate size and mass, and molecular dynamics (MD) simulations to model protein conformations and interactions. The order-of-addition experiments were conducted to examine the role of individual chaperones in the disaggregation and refolding process. Experiments were performed both at physiological temperatures and under heat-stress conditions to assess the equilibrium and non-equilibrium behavior of Hsp70.

The study found that different MLucV states (native, unfolded, aggregated, chaperone-bound) could be distinguished by FRET and luciferase activity. Urea or heat treatments produced small, soluble aggregates of approximately 12 MLucV monomers. Negative-stain electron microscopy confirmed the formation of discrete, roughly spherical particles with a narrow size distribution. SEC-RALS analysis showed an average mass consistent with a dodecamer. Molecular dynamics simulations supported a model where misfolded luciferase cores form the hydrophobic core of the aggregate, with the fluorescent domains positioned at the surface. Order-of-addition experiments showed that DnaJ is necessary for DnaK to bind and unfold the aggregates; DnaK then unfolds the substrate beyond its urea-unfolded conformation. GrpE promotes ADP/ATP exchange, leading to substrate release and refolding. At 38°C, where the native luciferase is unstable, the addition of the DnaK-DnaJ-GrpE system with ATP resulted in non-equilibrium accumulation of native MLucV. The system could actively target misfolding species, preventing their aggregation and allowing refolding, while not affecting native conformers. Experiments without ATP showed that the chaperones could not simply prevent aggregation by holding unfolded monomers; ATP hydrolysis is critical for the chaperones' activity. The study confirmed that Hsp70 acts as a stand-alone disaggregation machinery, independent of ClpB.

These findings provide unprecedented detail on the Hsp70 mechanism. The study demonstrates that Hsp70 does not simply prevent aggregation but actively disaggregates and unfolds stable protein aggregates. The involvement of DnaJ in targeting Hsp70 to misfolded substrates was clearly shown, along with the critical role of ATP in fueling this process. The results suggest a non-equilibrium mechanism where Hsp70 injects energy to unfold substrates beyond what is possible at equilibrium, allowing for refolding upon release. The findings have important implications for understanding protein quality control and the mechanisms that cells use to protect themselves against protein aggregation.

This study provides a detailed and comprehensive understanding of the Hsp70 unfolding mechanism using the novel MLucV reporter protein. The results demonstrate that Hsp70 acts as an ATP-fueled unfoldase, actively disaggregating and unfolding misfolded proteins, ultimately enabling their refolding into their native states. Future studies could investigate the mechanism in vivo using MLucV, explore the dynamics of individual chaperone interactions, and investigate the role of Hsp70 in more complex cellular contexts.

The study primarily used in vitro experiments with a bacterial Hsp70 system. While the use of MLucV allowed for a more homogenous system than typical protein aggregates, this system may not fully recapitulate the complexity of cellular environments. Further studies in vivo are required to fully validate these findings in a more complex cellular context. The molecular dynamics simulations, while helpful in visualizing protein interactions, are based on simplified models and may not capture all the details of the system’s dynamics.