•Recent structural insights in Hsp70 chaperones are reviewed.•Open and closed conformations as well as high- and low-affinity conformations are compared.•Influence of substrate binding on Hsp70 ...conformation and dynamics is presented.•Chaperone activities are discussed in the context of the dynamic nature of Hsp70s.
The chaperone functions of heat shock protein (Hsp)70 involve an allosteric control mechanism between the nucleotide-binding domain (NBD) and polypeptide substrate-binding domain (SBD): ATP binding and hydrolysis regulates the affinity for polypeptides, and polypeptide binding accelerates ATP hydrolysis. These data suggest that Hsp70s exist in at least two conformational states. Although structural information on the conformation with high affinity for polypeptides has been available for several years, the conformation with an open polypeptide binding cleft was elucidated only recently. In addition, other biophysical studies have revealed a more dynamic picture of Hsp70s, shedding light on the molecular mechanism by which Hsp70s assist protein folding. In this review recent insights into the structure and mechanism of Hsp70s are discussed.
Central to the chaperone function of Hsp70s is the transition between open and closed conformations of their polypeptide substrate binding domain (SBD), which is regulated through an allosteric ...mechanism via ATP binding and hydrolysis in their nucleotide binding domain (NBD). Although the structure of the closed conformation of Hsp70s is well studied, the open conformation has remained elusive. Here, we report on the 2.4 Å crystal structure of the ATP-bound open conformation of the Escherichia coli Hsp70 homolog DnaK. In the open DnaK structure, the β sheet and α-helical lid subdomains of the SBD are detached from one another and docked to different faces of the NBD. The contacts between the β sheet subdomain and the NBD reveal the mechanism of allosteric regulation. In addition, we demonstrate that docking of the β sheet and α-helical lid subdomains to the NBD is a sequential process influenced by peptide and protein substrates.
► Hsp70 structure in the ATP-bound open conformation reveals an allosteric mechanism ► Hsp70 structure suggests a catalytic mechanism for ATP hydrolysis ► Different parts of Hsp70 change conformation with different kinetics ► Bound substrate slows down the kinetics of conformational changes
The 70-kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that act in a large variety of cellular protein folding and remodelling processes. They function virtually at all stages of ...the life of proteins from synthesis to degradation and are thus crucial for maintaining protein homeostasis, with direct implications for human health. A large set of co-chaperones comprising J-domain proteins and nucleotide exchange factors regulate the ATPase cycle of Hsp70s, which is allosterically coupled to substrate binding and release. Moreover, Hsp70s cooperate with other cellular chaperone systems including Hsp90, Hsp60 chaperonins, small heat shock proteins and Hsp100 AAA+ disaggregases, together constituting a dynamic and functionally versatile network for protein folding, unfolding, regulation, targeting, aggregation and disaggregation, as well as degradation. In this Review we describe recent advances that have increased our understanding of the molecular mechanisms and working principles of the Hsp70 network. This knowledge showcases how the Hsp70 chaperone system controls diverse cellular functions, and offers new opportunities for the development of chemical compounds that modulate disease-related Hsp70 activities.
Hsp70 chaperones are central hubs of the protein quality control network and collaborate with co-chaperones having a J-domain (an ∼70-residue–long helical hairpin with a flexible loop and a conserved ...His–Pro–Asp motif required for ATP hydrolysis by Hsp70s) and also with nucleotide exchange factors to facilitate many protein-folding processes that (re)establish protein homeostasis. The Hsp70s are highly dynamic nanomachines that modulate the conformation of their substrate polypeptides by transiently binding to short, mostly hydrophobic stretches. This interaction is regulated by an intricate allosteric mechanism. The J-domain co-chaperones target Hsp70 to their polypeptide substrates, and the nucleotide exchange factors regulate the lifetime of the Hsp70–substrate complexes. Significant advances in recent years are beginning to unravel the molecular mechanism of this chaperone machine and how they treat their substrate proteins.
Molecular chaperones assist folding processes and conformational changes in many proteins. In order to do so, they progress through complex conformational cycles themselves. In this review, I discuss ...the diverse conformational dynamics of the ATP-dependent chaperones of the Hsp60, Hsp70, Hsp90, and Hsp100 families.
Efficient targeting of Hsp70 chaperones to substrate proteins depends on J-domain cochaperones, which in synergism with substrates trigger ATP hydrolysis in Hsp70s and concomitant substrate trapping. ...We present the crystal structure of the J-domain of Escherichia coli DnaJ in complex with the E. coli Hsp70 DnaK. The J-domain interacts not only with DnaK’s nucleotide-binding domain (NBD) but also with its substrate-binding domain (SBD) and packs against the highly conserved interdomain linker. Mutational replacement of contacts between J-domain and SBD strongly reduces the ability of substrates to stimulate ATP hydrolysis in the presence of DnaJ and compromises viability at heat shock temperatures. Our data demonstrate that the J-domain and the substrate do not deliver completely independent signals for ATP hydrolysis, but the J-domain, in addition to its direct influence on Hsp70s catalytic center, makes Hsp70 more responsive for the hydrolysis-inducing signal of the substrate, resulting in efficient substrate trapping.
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•We solved the crystal structure of a J-domain in complex with Hsp70·ATP•Our structure reveals how the J-domain stimulates Hsp70’s ATPase activity•The J-domain is linked to the catalytic ATPase center via a network of interactions•The J-domain is linked to residues that transmit the synergistic substrate signal
Kityk et al. determine the structure of the J-domain of a cochaperone in complex with Hsp70·ATP. Their work provides insights into how the J-domain and misfolded proteins collaborate to induce Hsp70 ATPase activity, allowing Hsp70 to trap the misfolded proteins.
Hsp90: Breaking the Symmetry Mayer, Matthias P.; Le Breton, Laura
Molecular cell,
04/2015, Volume:
58, Issue:
1
Journal Article
Peer reviewed
Open access
Hsp90 chaperones receive much attention due to their role in cancer and other pathological conditions, and a tremendous effort of many laboratories has contributed in the past decades to considerable ...progress in the understanding of their functions. Hsp90 chaperones exist as dimers and, with the help of cochaperones, promote the folding of numerous client proteins. Although the original view of these interactions suggested that these dimeric complexes were symmetrical, it is now clear that many features are asymmetrical. In this review we discuss several recent advances that highlight how asymmetric interactions with cochaperones as well as asymmetric posttranslational modifications provide mechanisms to regulate client interactions and the progression through Hsp90’s chaperone cycle.
Hsp90 chaperones receive much attention due to their role in cancer and other pathological conditions, and a tremendous effort of many laboratories has contributed in the past decades to considerable progress in the understanding of their functions. Hsp90 chaperones exist as dimers and, with the help of cochaperones, promote the folding of numerous client proteins. Although the original view of these interactions suggested that these dimeric complexes were symmetrical, it is now clear that many features are asymmetrical. In this review we discuss several recent advances that highlight how asymmetric interactions with cochaperones as well as asymmetric posttranslational modifications provide mechanisms to regulate client interactions and the progression through Hsp90’s chaperone cycle.
The 70 kDa heat-shock protein (Hsp70) is undoubtedly the most versatile of all molecular chaperones. Hsp70 is involved in numerous cellular protein folding processes, accompanying proteins throughout ...their lifespan from de novo folding at the ribosome to degradation at the proteasome, surveilling protein stability and functionality. Several properties of this ATP-dependent chaperone constitute the molecular basis for this versatility. With its substrate binding domain (SBD), Hsp70 transiently interacts with a short degenerative linear sequence motif found practically in all proteins and, in addition, with more folded protein conformers. Binding to polypeptides is tightly regulated by ATP binding and hydrolysis in the nucleotide binding domain, which is coupled to the SBD by an intricate allosteric mechanism. Hsp70 is regulated by a host of J-cochaperones, which act as targeting factors by regulating the ATPase activity of Hsp70 in synergism with the substrates themselves, and by several families of nucleotide exchange factors. In this review, I focus on the allosteric mechanism, which allows Hsp70s to interact with substrates with ultrahigh affinity through a non-equilibrium mode of action and summarize what mutagenesis and structural studies have taught us about the pathways and mechanics of interdomain communication.
This article is part of a discussion meeting issue ‘Allostery and molecular machines’.
The activity of the tumor suppressor p53 has to be timed and balanced closely to prevent untimely induction of cell death. The stability of p53 depends on the ubiquitin ligase Mdm2 but also on Hsp70 ...and Hsp90 chaperones that interact with its DNA binding domain (DBD). Using hydrogen exchange mass spectrometry and biochemical methods, we analyzed conformational states of wild-type p53-DBD at physiological temperatures and conformational perturbations in three frequent p53 cancer mutants. We demonstrate that the Hsp70/Hdj1 system shifts the conformational equilibrium of p53 toward a flexible, more mutant-like, DNA binding inactive state by binding to the DNA binding loop. The analyzed cancer mutants are likewise destabilized by interaction with the Hsp70/Hdj1 system. In contrast, Hsp90 protects the DBD of p53 wild-type and mutant proteins from unfolding. We propose that the Hsp70 and Hsp90 chaperone systems assume complementary functions to optimally balance conformational plasticity with conformational stability.
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•Hsp70/Hdj1 increase local unfolding in wild-type and mutant p53-DNA binding domain•Hsp70/Hdj1 dissociates p53 from DNA by binding to its DNA binding loop•Hsp90 counteracts unfolding by Hsp70/Hdj1 and globally stabilizes p53•Hsp70 and Hsp90 balance p53 transcriptional activity by regulating its conformation
The transcriptional activity of the tumor suppressor p53, which is essential to eliminate lethally damaged and oncogenic transformed cells, must be timed and balanced closely to prevent untimely induction of cell death. Boysen et al. show that Hsp70 and Hsp90 regulate p53’s conformation and DNA binding activity in opposing directions.
The heat shock transcription factors heat shock transcription factor 1 and Hsf2 have been studied for many years, mainly in the context of stress response and in malignant cells. Their physiological ...function in nonmalignant human cells under nonstress conditions is still largely unknown. To approach this important issue, Joutsen et al. present immunohistochemical staining data on Hsf1 and Hsf2 in 80 nonpathological human tissue samples. The wealth of these data elicits many interesting questions that will spur many future research projects.
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