(MEMPHIS, Tenn. – October 14, 2025) The first full-length structures of two heat shock chaperone proteins in a complex reveal the key structural region regulating their function, according to a new study from St. Jude Children's Research Hospital. The chaperone complex binds misfolded proteins and gives them another chance to fold correctly. This process is essential for cells because improperly folded proteins tend to aggregate, potentially causing a diverse array of diseases. However, the mechanism by which these two major heat shock chaperone proteins, Hsp40 and Hsp70, bind each other and misfolded peptides was unclear. The new structures enabled the scientists to identify how the cellular machinery works. The findings were published today in Molecular Cell.
"Heat shock chaperone proteins form the first line of defense against protein misfolding and aggregation, a precursor to many diseases," said corresponding author Charalampos Babis Kalodimos , PhD, St. Jude Department of Structural Biology chair. "We showed how these two particular heat shock chaperone proteins come together, revealing a previously unknown set of mechanisms explaining how they work in tandem, and how they can malfunction."
Heat shock chaperone proteins are essential in most organisms, with similar versions present from bacteria to humans. Bacterial Hsp40 and Hsp70 are known to help misfolded proteins correctly refold, with decades of research into their function as chaperone proteins. The human versions of these proteins are also linked to several diseases. However, exactly how Hsp40 and Hsp90 bind a client protein and each other was unknown, because no one had been able to overcome the technical hurdles involved in capturing the massive full-length structure.
Instead of using a single approach, the St. Jude scientists combined cryogenic electron microscopy, nuclear magnetic resonance imaging and X-ray crystallography data to finally solve the bacterial complex's structure. They captured multiple forms of the structure, consisting of dimers (pairs) of Hsp70 interacting with dimers of Hsp40, in several different states of binding client proteins.
"Most importantly, we captured the full-length structures of the active and inhibited states, bound to an incorrectly folded protein," Kalodimos said. "But we also solved multiple smaller pieces. By assembling them together, they've given us a new model of how the Hsp40 and Hsp70 complex really functions."
G/F region handles heat shock protein handoffs
Mutations in a specific region of Hsp40 are associated with an array of ailments, including many neurodegenerative diseases, but how those mutations disrupt normal functions was unknown. The region is rich in highly conserved amino acids called glycines and phenylalanines, though their exact functions were unclear. The structures revealed that this region of Hsp40 binds a client protein first, then a particular phenylalanine binds to the substrate binding site on Hsp70. That phenylalanine then pulls the misfolded protein into the substrate binding site, displacing itself, though the rest of Hsp40 remains bound to Hsp70. After the handoff, the misfolded peptide is then unfolded by Hsp70.
"We first uncovered the mechanism of handover, where Hsp40 gives the client to Hsp70 by initially tucking in a phenylalanine into the binding pocket, which subsequently transfers the client into the same site," Kalodimos said. "But then, by having structures of the complex in multiple states, we also saw how the complex releases client proteins."
The scientists found that when ATP, the energy currency of the cell, binds to Hsp70, Hsp70 changes shape, pushing the phenylalanine back into the binding pocket, which in turn pushes out the client protein and releases it into the cell. Afterward, Hsp40 leaves Hsp70 and is then available to bind another misfolded protein, starting the process again.
"We now understand mechanistically how the chaperones and protein client form this very big complex, how they are released, and how they cycle through these states," Kalodimos said. "Using that knowledge, we have a starting place to look for therapeutic interventions to compensate for disease-causing mutations, to exploit as drug targets in cancers or inspire next-generation antibiotics ."
Authors and funding
The study's co-first authors are Ziad Ibrahim and Youlin Xia, of St. Jude; and Yajun Jiang, Nanjing University. The study's other authors are Meixia Che, Nanjing University; Mary Clay, Kalyan Immadisetty, Zhilian Xia, Liang Tang, Paolo Rossi, Pritha Ganguly, Jiangshu Liu and Darcie Miller, St. Jude Children's Research Hospital; Alexandre Myasnikov, EPFL; and Santiago Palacios, Günter Kramer and Bernd Bukau, Heidelberg University.
The study was supported by grants from the National Institute of General Medical Sciences (P30GM133893), the U.S. Department of Energy Office of Biological and Environmental Research (KP1607011), the U.S. Department of Energy, Office of Science, through the National Synchrotron Light Source II (Contract No. DE-SC0012704), National Institutes of Health (R35 GM122462), the National Natural Science Foundation of China (32271316), the Jiangsu Provincial Science and Technology Major Plan Special Fund (BG2024026) and ALSAC, the fundraising and awareness organization of St. Jude.
