Phosphorylation of the Hsp90 Co-Chaperone Hop Changes its Conformational Dynamics and Biological Function

The molecular chaperones Hsp90 and Hsp70 and their regulatory co-chaperone Hop play a key role at the crossroads of the folding pathways of numerous client proteins by forming fine-tuned multiprotein complexes. Alterations of the biomolecules involved may functionally impact the chaperone machinery: here, we integrate simulations and experiments to unveil how Hop conformational fitness and interactions can be controlled by the perturbation of just one residue. Specifically, we unveil how mechanisms mediated by Hop residue Y354 control Hop open and closed states, which affect binding of Hsp70/Hsp90. Phosphorylation or mutation of Hop-Y354 are shown to favor structural ensembles that are indeed not optimal for stable interactions with Hsp90 and Hsp70. This disfavors cellular accumulation of the stringent Hsp90 clients glucocorticoid receptor and the viral tyrosine kinase v-Src, with detrimental effects on v-Src activity. Our results show how the post-translational modification of a specific residue in Hop provides a regulation mechanism for the larger chaperone complex of which it is part. In this framework, the effects of one single alteration are amplified at the cellular level through the perturbation of protein-interaction networks.     

Conformational dynamics of full-length inducible human Hsp70 derived from microsecond molecular dynamics simulations in explicit solvent

Human 70?kDa heat shock protein (hHsp70) is an ATP-dependent chaperone and is currently an important target for developing new drugs in cancer therapy. Knowledge of the conformations of hHsp70 is central to understand the interactions between its nucleotide-binding domain (NBD) and substrate-binding domain (SBD) and is a prerequisite to design inhibitors. The conformations of ADP-bound (or nucleotide-free) hHsp70 and ATP-bound hHsp70 was investigated by using unbiased all-atom molecular dynamics (MD) simulations of homology models of hHsp70 in explicit solvent on a timescale of .5 and 2.7 μs, respectively. The conformational heterogeneity of hHsp70 was analyzed by computing effective free-energy landscapes (FELs) and distance distribution between selected pair of residues. These theoretical data were compared with those extracted from single-molecule Förster resonance energy transfer (FRET) experiments and to small-angle X-ray scattering (SAXS) data of Hsp70 homologs. The distance between a pair of residues in FRET is systematically larger than the distance computed in MD which is interpreted as an effect of the size and of the dynamics of the fluorescent probes. The origin of the conformational heterogeneity of hHsp70 in the ATP-bound state is due to different binding modes of the helix B of the SBD onto the NBD. In the ADP-bound (or nucleotide-free) state, it arises from the different closed conformations of the SBD and from the different positions of the SBD relative to the NBD. In each nucleotide-binding state, Hsp70 is better represented by an ensemble of conformations on a μs timescale corresponding to different local minima of the FEL.

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NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems

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Conformational equilibria in allosteric control of Hsp70 chaperones

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NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperone

Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.     

Structural and functional analysis of the Hsp70/Hsp40 chaperone system

As one of the most abundant and highly conserved molecular chaperones, the 70‐kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.     

Real‐time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP‐hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate‐binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate‐ and nucleotide‐binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide‐free state. The nucleotide‐exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.     

Substrate relay in an Hsp70‐cochaperone cascade safeguards tail‐anchored membrane protein targeting

Membrane proteins are aggregation‐prone in aqueous environments, and their biogenesis poses acute challenges to cellular protein homeostasis. How the chaperone network effectively protects integral membrane proteins during their post‐translational targeting is not well understood. Here, biochemical reconstitutions showed that the yeast cytosolic Hsp70 is responsible for capturing newly synthesized tail‐anchored membrane proteins (TAs) in the soluble form. Moreover, direct interaction of Hsp70 with the cochaperone Sgt2 initiates a sequential series of TA relays to the dedicated TA targeting factor Get3. In contrast to direct loading of TAs to downstream chaperones, stepwise substrate loading via Hsp70 maintains the solubility and targeting competence of TAs, ensuring their efficient delivery to the endoplasmic reticulum (ER). Inactivation of cytosolic Hsp70 severely impairs TA translocation in vivo. Our results demonstrate a new role of cytosolic Hsp70 in directly assisting the targeting of an essential class of integral membrane proteins and provide a paradigm for how “substrate funneling” through a chaperone cascade preserves the conformational quality of nascent membrane proteins during their biogenesis.     

Common functionally important motions of the nucleotide‐binding domain of Hsp70

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate‐binding domain (SBD) that binds client substrates, and the nucleotide‐binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure–function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all‐atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP‐ and ATP‐unique classes, which reflect conformational trends that are unique to either the ADP‐ or ATP‐bound states, respectively. “Mutual” class motions generally describe “in‐plane” and/or “out‐of‐plane” (scissor‐like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The “unique” class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the “unique” type, regions of enhanced mobility can be identified; these are termed “hot spots,” and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide‐binding pocket was also found to influence the dynamics of the NBD significantly. Proteins 2015; 83:282–299. © 2014 Wiley Periodicals, Inc.     

Positive natural selection has driven the evolution of the Hsp70s in Diguetia spiders

Hsp70s are a ubiquitous family of highly conserved proteins. Hsp70s are chaperones and have important roles in both protein folding and thermotolerance. It has been widely assumed that Hsp70 sequence evolution is governed by the strong functional constraints imposed by its crucial cellular functions. In this study of cytosolic heat-inducible Hsp70s from three spider families, we have found clear evidence of positive natural selection altering Hsp70s in desert-dwelling and heat-loving Diguetidae spiders. These spiders are a small family restricted to deserts. They display heat-tolerant behaviours not seen in their closest relatives, the Pholcidae and Plectreuridae.     

Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Co‐evolution‐driven switch of J‐protein specificity towards an Hsp70 partner

Molecular mechanisms by which protein–protein interactions are preserved or lost after gene duplication are not understood. Taking advantage of the well–studied yeast mtHsp70:J–protein molecular chaperone system, we considered whether changes in partner proteins accompanied specialization of gene duplicates. Here, we report that existence of the Hsp70 Ssq1, which arose by duplication of the gene encoding multifunction mtHsp70 and specializes in iron–sulphur cluster biogenesis, correlates with functional and structural changes in the J domain of its J–protein partner Jac1. All species encoding this shorter alternative version of the J domain share a common ancestry, suggesting that all short JAC1 proteins arose from a single deletion event. Construction of a variant that extended the length of the J domain of a ‘short’ Jac1 enhanced its ability to partner with multifunctional Hsp70. Our data provide a causal link between changes in the J protein partner and specialization of duplicate Hsp70.     

Titration_DB: Storage and analysis of NMR‐monitored protein pH titration curves

NMR‐monitored pH titration experiments are routinely used to measure site‐specific protein pKa values. Accurate experimental pKa values are essential in dissecting enzyme catalysis, in studying the pH‐dependence of protein stability and ligand binding, in benchmarking pKa prediction algorithms, and ultimately in understanding electrostatic effects in proteins. However, due to the complex ways in which pH‐dependent electrostatic and structural changes manifest themselves in NMR spectra, reported apparent pKa values are often dependent on the way that NMR pH‐titration curves are analyzed. It is therefore important to retain the raw NMR spectroscopic data to allow for documentation and possible re‐interpretation. We have constructed a database of primary NMR pH‐titration data, which is accessible via a web interface. Here, we report statistics of the database contents and analyze the data with a global perspective to provide guidelines on best practice for fitting NMR titration curves. Titration_DB is available at http://enzyme.ucd.ie/Titration_DB . Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Rapamycin conditionally inhibits Hsp90 but not Hsp70 mRNA translation in <Emphasis Type="Italic">Drosophila</Emphasis>: implications for the mechanisms of Hsp mRNA translation

Rapamycin inhibits the activity of the target of rapamycin (TOR)-dependent signaling pathway, which has been characterized as one dedicated to translational regulation through modulating cap-dependent translation, involving eIF4E binding protein (eIF4E-BP) or 4E-BP. Results show that rapamycin strongly inhibits global translation in Drosophila cells. However, Hsp70 mRNA translation is virtually unaffected by rapamycin treatment, whereas Hsp90 mRNA translation is strongly inhibited, at normal growth temperature. Intriguingly, during heat shock Hsp90 mRNA becomes significantly less sensitive to rapamycin-mediated inhibition, suggesting the pathway for Hsp90 mRNA translation is altered during heat shock. Reporter mRNAs containing the Hsp90 or Hsp70 mRNAs’ 5′ untranslated region recapitulate these rapamycin-dependent translational characteristics, indicating this region regulates rapamycin-dependent translational sensitivity as well as heat shock preferential translation. Surprisingly, rapamycin-mediated inhibition of Hsp90 mRNA translation at normal growth temperature is not caused by 4E-BP-mediated inhibition of cap-dependent translation. Indeed, no evidence for rapamycin-mediated impaired eIF4E function is observed. These results support the proposal that preferential translation of different Hsp mRNA utilizes distinct translation mechanisms, even within a single species.     

Osmolyte‐induced conformational changes in the Hsp90 molecular chaperone

Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native‐state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X‐ray scattering we observe dramatic osmolyte‐dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well‐established osmolyte principles that govern stability also apply to large‐scale conformational changes, a proposition that is corroborated by structure‐based fitting of the scattering data, surface area comparisons and m‐value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain‐domain interaction. Hsp90 adopts an additional ligand‐specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.     

Structure and function of the molecular chaperone Hsp104 from yeast

The molecular chaperone Hsp104 plays a central role in the clearance of aggregates after heat shock and the propagation of yeast prions. Hsp104's disaggregation activity and prion propagation have been linked to its ability to resolubilize or remodel protein aggregates. However, Hsp104 has also the capacity to catalyze protein aggregation of some substrates at specific conditions. Hence, it is a molecular chaperone with two opposing activities with respect to protein aggregation. In yeast models of Huntington's disease, Hsp104 is required for the aggregation and toxicity of polyglutamine (polyQ), but the expression of Hsp104 in cellular and animal models of Huntington's and Parkinson's disease protects against polyQ and α‐synuclein toxicity. Therefore, elucidating the molecular determinants and mechanisms underlying the ability of Hsp104 to switch between these two activities is of critical importance for understanding its function and could provide insight into novel strategies aimed at preventing or reversing the formation of toxic protein aggregation in systemic and neurodegenerative protein misfolding diseases. Here, we present an overview of the current molecular models and hypotheses that have been proposed to explain the role of Hsp104 in modulating protein aggregation and prion propagation. The experimental approaches and the evidences presented so far in relation to these models are examined. Our primary objective is to offer a critical review that will inspire the use of novel techniques and the design of new experiments to proceed towards a qualitative and quantitative understanding of the molecular mechanisms underlying the multifunctional properties of Hsp104 in vivo. © 2009 Wiley Periodicals, Inc. Biopolymers 93:252–276, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Hsp70 binds to PrPC in the process of PrPC release via exosomes from THP-1 monocytes

PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein present on the surface of a number of peripheral blood cells. PrPC must be present for the generation and propagation of pathogenic conformer [PrPSc (scrapie prion protein)], which is a conformational conversion form of PrPC and has a central role in transmissible spongiform encephalopathies. It is important to determine the transportation mechanism of normal PrPC between cells. Exosomes are membrane vesicles released into the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. We have identified that THP-1 monocytes can secrete exosomes to culture medium, and the secreted exosomes can bear PrPC. We also found that Hsp70 interacts with PrPC not only in intracellular environment, but in the secreted exosomes. However, the specific markers of exosomes, Tsg101 and flotillin-1, were found with no interaction with PrPC. Our results demonstrated that PrPC can be released from THP-1 monocytes via secreted exosomes, and in this process, Hsp70 binds to PrPC, which suggests that Hsp70 may play a potential functional role in the release of PrPC.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

Mild heat stress at a young age in<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> leads to increased Hsp70 synthesis after stress exposure later in life

In a number of animal species it has been shown that exposure to low levels of stress at a young age has a positive effect on stress resistance later in life, and on longevity. The positive effects have been attributed to the activation of defence/cleaning systems (heat shock proteins (Hsps), antioxidases, DNA repair) or to effects of a changed metabolic rate, or both. We investigated the effect of mild stress exposures early in life on Hsp70 synthesis after a harder stress exposure later in life in five isofemale lines ofDrosophila melanogaster. Female flies were either exposed to repeated bouts of mild heat stress (3 h at 34‡C) at a young age (days 2, 4 and 6 post-eclosion) or held under standard laboratory conditions. At 16 and 32 days of adult age, respectively, flies were exposed to a high temperature treatment known to induce Hsp70 in the investigated species (1 h at 37‡C). Thereafter, the inducible Hsp70 levels were measured. Our data show a tendency towards increased Hsp70 synthesis with increased age for both ’mild stress’ and ’no stress’ flies. Moreover, the results show that flies exposed to mild stress at a young age synthesized more Hsp70 upon induction, compared to control flies, and that this difference was accentuated at 32 days compared to 16 days of age. Thus, bouts of mild heat stress at a young age impact on the physiological stress response system later in life. This may be caused by an increased ability to react to future stresses. Alternatively, the mild stress exposure at a young age may actually have caused cellular damages increasing the need for Hsp70 levels after stress exposure later in life. The importance of an Hsp70 upregulation (throughout life) in explaining the phenomenon of hormesis is discussed, together with alternative hypotheses, and suggestions for further studies.