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Hsp90 is an essential and highly conserved modular molecular chaperone whose N and middle domains are separated by a disordered region termed the charged linker. Although its importance has been previously disregarded, because a minimal linker length is sufficient for Hsp90 activity, the evolutionary persistence of extensive charged linkers of divergent sequence in Hsp90 proteins of most eukaryotes remains unexplained. To examine this question further, we introduced human and plasmodium native and length-matched artificial linkers into yeast Hsp90. After evaluating ATPase activity and biophysical characteristics in vitro, and chaperone function in vivo, we conclude that linker sequence affects Hsp90 function, cochaperone interaction, and conformation. We propose that the charged linker, in addition to providing the flexibility necessary for Hsp90 domain rearrangements-likely its original purpose-has evolved in eukaryotes to serve as a rheostat for the Hsp90 chaperone machine. [ABSTRACT FROM AUTHOR]

Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Among the ATP-dependent chaperones, heat shock proteins (Hsp90) proteins play a special role. While Hsp90s can interact with unfolded and misfolded proteins, their main (and in eukaryotic cells essential) function appears to involve interactions with a limited number of protein clients at late steps of maturation or in 'alter-native' conformations for regulating their stability and activity. Because Hsp90 clients are hubs of diverse signaling networks and participate in nearly every cellular function, Hsp90s interconnect many regulatory circuits and link them to environmental impacts. The availability and activity of Hsp90 may thus influence complex physiological and pathophysiological processes, such as differentiation, development, aging, cancer, neurodegeneration, and infectious diseases. Furthermore, through homeostatic effects on differentiation and development, Hsp90s act as capacitors of phenotypic evolution. In this review, we discuss recent insights in the structure and chaperone cycle of Hsp90s, the mechanisms underlying Hsp90 binding to clients, and potential reasons why client proteins specifically require the assistance of Hsp90s. Moreover, the current views on Hsp90-cochaperone interactions and regulation of Hsp90 proteins via posttranslational modifications are summarized. The second half of this article is devoted to the role of Hsp90 proteins in health and disease, aging, and evolution. [ABSTRACT FROM AUTHOR]

Abstract: Optical fiber sensors based on intensity measurement require some form of intensity referencing to avoid errors arising from parasitic losses. Known techniques of referencing such as balanced bridge, divided beam systems or two-wavelength referencing are not suitable for low-cost applications since they use relatively complicated optical components such as multiple LED sources, couplers, filters etc. In this work a novel method of referencing in an extrinsic optical fiber sensor system utilizing temperature dependence of absorption edge in a semiconductor crystal is described. The sensor system comprises a single LED source and no optical fiber junctions. The emission spectrum of an LED depends on its temperature. The reference is provided by controlling the temperature of an LED source and transmission measurements with different emission spectra. The entire process is controlled by a microprocessor unit. Performance of a sensor system is investigated and it is shown that the losses in connectors may be compensated for. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The highly conserved 90 kDa heat shock protein (Hsp90) chaperones use ATP to regulate the stability and activity of many signalling molecules like protein kinases and transcription factors. Studies using crystallography, electron microscopy and small-angle X-ray scattering yielded controversial results for the conformational states that these dimeric multidomain proteins assume while progressing through the ATPase cycle. To better understand the molecular mechanism of Hsp90 proteins, we studied the conformational dynamics of the Escherichia coli homologue HtpG in solution using amide hydrogen exchange mass spectrometry (HX-MS) and fluorescence spectroscopy. A conformation-sensitive fluorescent probe allowed to elucidate the ATPase cycle of HtpG. Continuous-labelling and pulse-labelling HX-MS experiments revealed major ATP-induced conformational changes throughout the protein that do not occur simultaneously, but progress surprisingly slow from the immediate nucleotide-binding site towards the N terminus and the middle domain. The conversion between the different conformational states is rate limiting for ATP hydrolysis, and the nucleotide-coordinating residue, Glu34, is important for the rate constant of conversion. Our findings, for the first time, allow to kinetically resolve changes in the conformational dynamics of individual structural elements of Hsp90. [ABSTRACT FROM AUTHOR]

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones. [ABSTRACT FROM AUTHOR]

The Hsp70 chaperone activity in protein folding is regulated by ATP-controlled cycles of substrate binding and release. Nucleotide exchange plays a key role in these cycles by triggering substrate release. Structural searches of Hsp70 homologs revealed three structural elements within the ATPase domain: two salt bridges and an exposed loop. Mutational analysis showed that these elements control the dissociation of nucleotides, the interaction with exchange factors and chaperone activity. Sequence variations in the three elements classify the Hsp70 family members into three subfamilies, DnaK proteins, HscA proteins and Hsc70 proteins. These subfamilies show strong differences in nucleotide dissociation and interaction with the exchange factors GrpE and Bag-1. [ABSTRACT FROM AUTHOR]

The 70 kDa heat shock proteins (the Hsp70 family) assist refolding of their substrates through ATP-controlled binding. We have analyzed mutants of DnaK, an Hsp70 homolog, altered in key residues of its substrate binding domain. Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered closing device involving independent action of an α-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a β-structured subdomain. Correlative evidence from mutational analysis suggests that the ADP and ATP states of DnaK differ in the frequency of the conformational changes in the α-helical lid and β-domain that cause opening of the substrate binding cavity. The affinity for substrates, as defined by this mechanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis mediated locking-in of substrates and chaperone activity of DnaK. [ABSTRACT FROM AUTHOR]

Investigates the features of the mechanism by which DnaJ cooperates with Hsp70 chaperones, using Escherichia coli DnaJ and DnaK cochaperones as a model. Interaction between DnaJ and DnaK chaperones; How mutations in the substrate-binding pocket of DnaK affect stimulation of ATPase activity by DnaJ; How mutations in DnaK altering the interdomain communication affect stimulation of ATPase activity by DnaJ.

The desaturation of phytoene to Ζ-carotene and of Ζ-carotene to lycopene employs molecular oxygen as the terminal electron acceptor. 2,3,5,6-Tetramethyl-1,4-benzoquinone (duroquinone) and other artificial quinones are able to replace oxygen, which demonstrates that oxygen does not act in a mixed-function oxygenase-like mechanism at the desaturase itself, but at a spatially separate site. Evidence for additional red ox elements mediating between desaturase and oxygen is presented. [ABSTRACT FROM AUTHOR]

The σE (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The σE regulon is induced upon misfolding of proteins in the periplasm or the outer membrane. Similar to other alternative sigma factors, the activity of σE is tightly regulated in E. coli. We have previously shown that σE is positively autoregulated at the transcriptional level. DNA sequencing, coupled with transcriptional analyses, have shown that σE is encoded by the first gene of a four-gene operon. The second gene of this operon, rseA, encodes an anti-σE activity. This was demonstrated at both the genetic and biochemical levels. For example, mutations in rseA constitutively increase σE activity. Consistent with this, overproduction of RseA leads to an inhibitory effect on σE activity. Topological analysis of RseA suggests the existence of one transmembrane domain, with the N-terminal part localized in the cytoplasm. Overproduction of this N-terminal domain alone was shown to inhibit σE activity. These observations were confirmed in vitro, because either purified RseA or only its purified N-terminal domain inhibited transcription from EσE-dependent promoters. Furthermore, RseA and σE co-purify, and can be co-immunoprecipitated, and chemically cross-linked. The σE activity is further modulated by the products of the remaining genes in this operon, rseB and rseC. RseB is a periplasmic protein, which negatively regulates σE activity and specifically interacts with the C-terminal periplasmic domain of RseA. In contrast, RseC is an inner membrane protein that positively modulates σE activity. Most of these protein-protein interactions were verified in vivo using the yeast two-hybrid system. [ABSTRACT FROM AUTHOR]

The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of Hsf1 activity regulation remains poorly understood at the molecular level. In metazoa, Hsf1 trimerizes upon heat shock through a leucine‐zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, using purified proteins, we demonstrate that unmodified trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 binds to multiple sites in Hsf1 with different affinities. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucine‐zipper. Starting this unzipping at several protomers of the Hsf1 trimer results in faster monomerization. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation, Hsc70 first binds to a high‐affinity site in the transactivation domain, leading to partial attenuation of the response, and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state. Synopsis: Heat shock transcription factor 1 (Hsf1) is a central regulator of the heat shock response (HSR) in eukaryotic cells. This study demonstrates that the Hsp70 system attenuates the metazoa HSR and restores the resting state, by monomerizing trimeric Hsf1 and thus dissociating it from DNA. Hsp70 chaperones together with J‐domain co‐chaperones dissociate Hsf1 from DNA in vitro.Hsf1 is bound by Hsp70s at distinct sites with different affinities, allowing Hsf1 to closely monitor cellular Hsp70 levels.Starting at the binding site adjacent to the trimerization domain, Hsp70s monomerize Hsf1 trimers by successive cycles of entropic pulling.Mutation or deletion of the identified binding sites potentiate Hsf1 activity in cell culture models. [ABSTRACT FROM AUTHOR]

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware. [ABSTRACT FROM AUTHOR]

[Display omitted] • Y/E and Y/F exchanges in hHsp90α differentially influence client activity in yeast. • Y/E and Y/F exchanges in Hsp90 modulate conformation and affinity for cochaperones. • Multiple Y/E and Y/F replacements do not act additive but sometimes compensatory. • Our data suggest that PTMs bias human Hsp90s in their client selection. The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90β, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1 −/− cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity. [ABSTRACT FROM AUTHOR]

Abstract: The 90‐kDa heat shock proteins [heat shock protein 90 (Hsp90)] are a highly conserved ATP-dependent protein family, which can be found from prokaryotic to eukaryotic organisms. In general, Hsp90s are elongated dimers with N- and C-terminal dimerization sites. In a series of publications, we have recently shown that no successive mechanochemical cycle exists for yeast Hsp90 (yHsp90) in the absence of clients or cochaperones. Here, we resolve the mechanochemical cycle of the bacterial homologue HtpG by means of two‐ and three‐color single‐molecule FRET (Förster resonance energy transfer). Unlike yHsp90, the N-terminal dynamics of HtpG is strongly influenced by nucleotide binding and turnover—its reaction cycle is driven by a mechanical ratchet mechanism. However, the C-terminal dimerization site is mainly closed and not influenced by nucleotides. The direct comparison of both proteins shows that the Hsp90 machinery has developed to a more flexible and less nucleotide-controlled system during evolution. [Copyright &y& Elsevier]

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis. Author summary: To survive in the human body, the malaria parasite invades and lives within human red blood cells. Once within the red blood cell, the parasite renovates the host cell to its own needs. Here we have studied which factors from both parasite and host cell are required for this renovation process, and discover that human chaperone proteins, referred to as HSP70, are required. It appears that a particular parasite-derived protein, PFA66, recruits and modifies the function of the human HSP70. As this interaction between a parasite and human protein is novel and essential for parasite survival, our study identifies a potential Achilles' Heel which may be targeted for development of new anti-malaria therapies. [ABSTRACT FROM AUTHOR]

The heat shock response is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Post-translational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells, and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by small ubiquitin-like modifier (SUMO) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low-level SUMOylation at other sites. Different SUMO E3 ligases such as protein inhibitor of activated STAT 4 enhanced the efficiency of in vitro modification but did not alter SUMO site preferences. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA bound, and SUMOylation of Hsf1 does neither alter DNA-binding affinity nor affects heat shock cognate 71kDa protein (HSPA8)+DnaJ homolog subfamily B member 1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the heat shock response. [ABSTRACT FROM AUTHOR]

Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. HumanHSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of cochaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s,HSPA1Aand HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis- associated protein variant superoxide dismutase 1 (SOD1)- A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factorHSPH2preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2. [ABSTRACT FROM AUTHOR]

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. • 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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

Chaperones assist in protein folding, but what this common phrase means in concrete terms has remained surprisingly poorly understood. We can readily measure chaperone binding to unfolded proteins, but how they bind and affect proteins along folding trajectories has remained obscure. Here we review recent efforts by our labs and others that are beginning to pry into this issue, with a focus on the chaperones trigger factor and Hsp70. Single-molecule methods are central, as they allow the stepwise process of folding to be followed directly. First results have already revealed contrasts with long-standing paradigms: rather than acting only “early” by stabilizing unfolded chain segments, these chaperones can bind and stabilize partially folded structures as they grow to their native state. The findings suggest a fundamental redefinition of the protein folding problem and a more extensive functional repertoire of chaperones than previously assumed. [ABSTRACT FROM AUTHOR]

The 90-kDa heat shock protein (Hsp90) chaperones the late folding steps of many protein kinases, transcription factors, and a diverse set of other protein clients not related in sequence and structure. Hsp90's interaction with clients appears to be coupled to a series of conformational changes. How these conformational changes contribute to its chaperone activity is currently unclear. Using crosslinking, hydrogen exchange mass spectrometry, and fluorescence experiments, we demonstrate here that the N-terminal domain of Hsp90 rotates by approximately 180° as compared to the crystal structure of yeast Hsp90 in complex with Sba1 and AMPPNP. Surprisingly, Aha1 but not Sba1 suppresses this rotation in the presence of AMPPNP but not in its absence. A minimum length of the largely unstructured linker between N-terminal and middle domain is necessary for this rotation, and interfering with the rotation strongly affects the interaction with Aha1 and the intrinsic and Aha1-stimulated ATPase activity. Surprisingly, suppression of the rotation only affects the activity of some clients and does not compromise yeast viability. [ABSTRACT FROM AUTHOR]

The 90-kDa heat shock proteins (Hsp90s) assist the maturation of many key regulators of signal transduction pathways and cellular control circuits like protein kinases and transcription factors and chaperone their stability and activity. In this function, Hsp90s cooperate with some 30 cochaperones and they are themselves subject to regulation by numerous post-translational modifications. In vertebrates, two major isoforms exist in the cytosol, Hsp90α and Hsp90β, which share a high degree of sequence identity and are expressed in tissue- and environmental condition-dependent manner. We identified an isoform-specific phosphorylation site in human Hsp90β. This phosphorylation site seems to be linked to vertebrate evolution since it is not found in invertebrata but in all tetrapoda and many but not all fish species. We provide data suggesting that this phosphorylation is important for the activation of Hsp90 clients like glucocorticoid receptor and a protein kinase. Replacement of the phosphorylation site by glutamate affects the conformational dynamics of Hsp90 and interaction with the kinase-specific cochaperone Cdc37. [ABSTRACT FROM AUTHOR]