The switch from client holding to folding in the Hsp70/Hsp90 chaperone machineries is regulated by a direct interplay between co-chaperones.

Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here, we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfold a stringent client, the glucocorticoid receptor ligand-binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. Current concepts assume that to proceed to client folding, Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 functionally interacts with Hop, relieves the stalling Hsp90-Hop interaction, and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle.

Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries.

p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client.

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2.

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.

The Plasticity of the Hsp90 Co-chaperone System.

The Hsp90 system in the eukaryotic cytosol is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function. Although progress has been made regarding the underlying biochemical mechanisms, how co-chaperones influence Hsp90 client proteins in vivo has remained elusive. By investigating the effect of 12 Hsp90 co-chaperones on the activity of different client proteins in yeast, we find that deletion of co-chaperones can have a neutral or negative effect on client activity but can also lead to more active clients. Only a few co-chaperones are active on all clients studied. Closely related clients and even point mutants can depend on different co-chaperones. These effects are direct because differences in client-co-chaperone interactions can be reconstituted in vitro. Interestingly, some co-chaperones affect client conformation in vivo. Thus, co-chaperones adapt the Hsp90 cycle to the requirements of the client proteins, ensuring optimal activation.

Active unfolding of the glucocorticoid receptor by the Hsp70/Hsp40 chaperone system in single-molecule mechanical experiments.

The glucocorticoid receptor (GR) is an important transcription factor and drug target linked to a variety of biological functions and diseases. It is one of the most stringent physiological clients of the Hsp90/Hsp70/Hsp40 chaperone system. In this study, we used single-molecule force spectroscopy by optical tweezers to observe the interaction of the GR's ligand-binding domain (GR-LBD) with the Hsp70/Hsp40 chaperone system (Hsp70/40). We show in real time that Hsp70/40 can unfold the complete GR-LBD in a stepwise manner. Each unfolding step involves binding of an Hsp70 to the GR-LBD and subsequent adenosine triphosphate (ATP) hydrolysis, stimulated by Hsp40. The kinetics of chaperone-mediated unfolding depend on chaperone concentrations as well as the presence of the nucleotide exchange factor BAG1. We find that Hsp70/40 can stabilize new unfolding intermediates, showing that Hsp70/40 can directly interact with the folded core of the protein when working as an unfoldase. Our results support an unfolding mechanism where Hsp70 can directly bind to folded protein structures and unfold them upon ATP hydrolysis. These results provide important insights into the regulation of GR by Hsp70/40.

Modulation of the Hsp90 chaperone cycle by a stringent client protein.

Hsp90 is the most abundant molecular chaperone in the eukaryotic cell. One of the most stringent clients is the glucocorticoid receptor (GR), whose in vivo function strictly depends on the interaction with the Hsp90 machinery. However, the molecular mechanism of this interaction has been elusive. Here we have reconstituted the interaction of Hsp90 with hormone-bound GR using purified components. Our biochemical and structural analyses define the binding site for GR on Hsp90 and reveal that binding of GR modulates the conformational cycle of Hsp90. FRET experiments demonstrate that a partially closed form of the Hsp90 dimer is the preferred conformation for interaction. Consistent with this, the conformational cycle of Hsp90 is decelerated, and its ATPase activity decreases. Hsp90 cochaperones differentially affect formation of the Hsp90-GR complex, serving as control elements for cycle progression and revealing an intricate interplay of client and cochaperones as molecular modulators of the Hsp90 machine.

Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor.

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a "lid" structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of [Formula: see text] The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional [Formula: see text] of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s-1 From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

Nematode Sgt1-homologue D1054.3 binds open and closed conformations of Hsp90 via distinct binding sites.

Heat shock protein 90 (Hsp90) is a highly conserved ATP-driven machine involved in client protein maturation, folding, and activation. The chaperone is supported by a set of cochaperones that confer client specificities. One of those proteins is the suppressor of G2 allele of skp1 (Sgt1), which participates together with Hsp90 in the immune responses of plants. Sgt1 consists of three domains: a TPR-, CS-, and SGS-domain, conserved in plants, yeast, and humans. The TPR-domain though is lacking in nematodes and insects. We observe that the Caenorhabditis elegans Sgt1 homologue D1054.3 binds to Hsp90 in the absence of nucleotides but much stronger in the presence of ATP and ATPγS. The latter binding mode is similar to p23, another CS-domain containing Hsp90 cofactor, even though binding is not observable for p23 in the absence of nucleotides. We use point mutations in Hsp90, which accumulate different conformations in the ATPase cycle, to differentiate between binding to open and closed Hsp90 conformations. These data support a strong contribution of the Hsp90 conformation to Sgt1 binding and highlight the ability of this cofactor to interact with all known Hsp90 conformations albeit with different affinities.

Cdc37 (cell division cycle 37) restricts Hsp90 (heat shock protein 90) motility by interaction with N-terminal and middle domain binding sites.

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.

Cost comparison analysis of open versus laparoscopic distal pancreatectomy.

BACKGROUND: In comparison with open distal pancreatectomy (ODP), laparoscopic distal pancreatectomy (LDP) is associated with fewer complications and shorter hospital stays, but comparative cost data for the two approaches are limited. METHODS: Records of all distal pancreatectomies carried out from January 2009 to June 2013 were reviewed and stratified according to operative complexity. Patient factors and outcomes were recorded. Total variable costs (TVCs) were tabulated for each patient, and stratified by category [e.g. 'floor', 'operating room' (OR), 'radiology']. Costs for index admissions and 30-day readmissions were compared between LDP and ODP groups. RESULTS: Of 153 procedures, 115 (70 LDP, 45 ODP) were selected for analysis. The TVC of the index admission was US$3420 less per patient in the LDP group (US$10 480 versus US$13 900; P = 0.06). Although OR costs were significantly greater in the LDP cohort (US$5756 versus US$4900; P = 0.02), the shorter average hospitalization in the LDP group (5.2 days versus 7.7 days; P = 0.01) resulted in a lower overall cost. The total cost of index hospitalization combined with readmission was significantly lower in the LDP cohort (US$11 106 versus US$14 803; P = 0.05). CONCLUSIONS: In appropriately selected patients, LDP is more cost-effective than ODP. The increased OR cost associated with LDP is offset by the shorter hospitalization. These data clarify targets for further cost reductions.

Early identification of patients at increased risk for hepatic insufficiency, complications and mortality after major hepatectomy.

OBJECTIVE: Total bilirubin (TB) of >7 mg/dl is an accepted definition of postoperative hepatic insufficiency (PHI) given its association with the occurrence of complications and mortality after hepatectomy. The aim of this study was to identify a surrogate marker for PHI early in the postoperative course. METHODS: A single-institution database of patients undergoing major hepatectomy (three or more segments) during 2000-2012 was retrospectively reviewed. Demographic, clinicopathologic and perioperative factors were assessed for their association with PHI, defined as postoperative TB of >7 mg/dl or new ascites. Secondary outcomes included complications, major complications (Clavien-Dindo Grades III-V) and 90-day mortality. RESULTS: A total of 607 patients undergoing major hepatectomy without bile duct reconstruction were identified. Postoperative hepatic insufficiency occurred in 60 (9.9%) patients. A postoperative day 3 (PoD 3) TB level of ≥3 mg/dl was the only early perioperative factor associated with the development of PHI on multivariate analysis [hazard ratio (HR) = 7.81, 95% confidence interval (CI) 3.74-16.31; P < 0.001]. A PoD 3 TB of ≥3 mg/dl was associated with increased risk for postoperative complications (75.7% versus 53.9%), major complications (45.6% versus 17.6%), and 90-day mortality (15.5% versus 2.3%). This association persisted on multivariate analysis for any complications (HR = 1.98, 95% CI 1.10-3.54; P = 0.022), major complications (HR = 3.18, 95% CI 1.90-5.32; P < 0.001), and 90-day mortality (HR = 8.11, 95% CI 3.00-21.92; P < 0.001). CONCLUSIONS: Total bilirubin of ≥3 mg/dl on PoD 3 after major hepatectomy is associated with PHI, increased complications, major complications and 90-day mortality. This marker may serve as an early postoperative predictor of hepatic insufficiency.

The Optimal Amount of Lithium Difluorophosphate as an Additive for Si-Dominant Anodes in an Application-Oriented Setup.

Fluoroethylene carbonate (FEC) and vinylene carbonate (VC) are considered the most effective electrolyte additives for improving the solid electrolyte interphase (SEI) of Si-containing anodes while lithium difluorophosphate (LiDFP) is known to improve the interphases of cathode materials and graphite. Here, we combine VC, FEC, and different amounts of LiDFP in a highly-concentrated electrolyte to investigate the effect on Si-dominant anodes in detail. Cycle life tests, electrochemical impedance spectroscopy and rate tests with anode potential monitoring were conducted in Si/NCM pouch cells. The results reveal that adding LiDFP to the electrolyte improves all performance criteria of the full cells, with a concentration of 1 wt% being the optimal value for most cases. Post-mortem analyses using scanning electron microscopy and x-ray photoelectron spectroscopy showed that a more beneficial SEI film was formed for higher LiDFP concentrations, which led to less decomposition of electrolyte components and a better-maintained anode microstructure.

Acute Chest Syndrome.

Audience: Emergency medicine residents and medical students on emergency medicine rotations. Introduction: Acute chest syndrome is a life-threatening, potentially catastrophic complication of sickle cell disease.1,2 It occurs in approximately 50% of patients with sickle cell disease, with up to 13% all-cause mortality.1 Most common in children aged 2-4, up to 80% of patients with a prior diagnosis of acute chest syndrome will have recurrence of this syndrome.4 Diagnostic criteria include a new infiltrate on pulmonary imaging combined with any of the following: fever > 38.5°C (101.3°F), cough, wheezing, hypoxemia (PaO2 < 60 mm Hg), tachypnea, or chest pain.4,5 The pathophysiology of acute chest syndrome involves vaso-occlusion in pulmonary vessels resulting in hypoxia, release of inflammatory mediators, acidosis, and infarction of lung tissue. The most common precipitants are infections (viral or bacterial), rib infarction, and fat emboli.1,2,4 Patients commonly present with fever, dyspnea, cough, chills, chest pain, or hemoptysis. Diagnosis is made through physical exam, blood work, and chest imaging.1,2 Chest radiograph is considered the gold standard for imaging modality.3 Management of acute chest syndrome includes hydration with IV crystalloid solutions, antibiotics, judicious analgesia, oxygen, and, in severe cases, transfusion.6 Emergency medicine practitioners should keep acute chest syndrome as a cannot miss, high consequence differential diagnosis for all patients with sickle cell disease presenting to the Emergency Department. Educational Objectives: At the end of this oral board session, examinees will: 1) demonstrate the ability to obtain a complete medical history; 2) demonstrate the ability to perform a detailed physical examination in a patient with respiratory distress; 3) identify a patient with respiratory distress and hypoxia and manage appropriately (administer oxygen, place patient on monitor); 4) investigate the broad differential diagnoses which include acute chest syndrome, pneumonia, acute coronary syndrome, acute congestive heart failure, acute aortic dissection and acute pulmonary embolism; 5) list the appropriate laboratory and imaging studies to differentiate acute chest syndrome from other diagnoses (complete blood count, comprehensive metabolic panel, brain natriuretic peptide (BNP), lactic acid, procalcitonin, EKG, troponin level, d-dimer, chest radiograph); 6) identify a patient with acute chest syndrome and manage appropriately (administer intravenous pain medications, administer antibiotics after obtaining blood cultures, emergent consultation with hematology) and 7) provide appropriate disposition to the intensive care unit after consultation with hematology. Educational Methods: This case is used as a method to assess learners' ability to rapidly assess a patient in respiratory distress. The learner needs to address a limited differential diagnosis list while simultaneously stabilizing and treating the patient. The "patient" becomes an active participant in the case, with repeated requests for pain medication, and appropriate analgesic administration is required as a critical action. For faculty, this case is used to assist with periodic assessment of resident performance while in the emergency department (ED).We use oral board testing as one additional tool to assess residents' critical thinking, while still applying the pressure that is needed to pass the oral certification examination. Large groups of residents can be assessed in short periods of time without needing to "wait" for this particular patient presentation to be seen in the ED.In this case, learners were assessed using a free online evaluation tool, Google forms. Multiple questions were written for each critical action, and the Google form served as the online evaluation and repository of this information. The critical actions of the case were then tied to Emergency Medicine Milestones, and the results were compiled for use during resident clinical competency evaluations. Residents were provided with immediate feedback of their performance and were also given their electronic evaluations when requested. Research Methods: To assess the strengths and weaknesses of the case, learners and instructors were given the opportunity to provide electronic feedback after the case was completed. Subsequent modifications were made based on the feedback provided. Additionally, learners answered written multiple-choice questions after the case to assess for retention of the material. Results: Senior and junior residents alike enjoyed the process of an oral board simulation as an alternative to a more formal lecture. Seniors also stated that they felt more confident with their ability to pass the oral certification examination after having gone through oral board testing while in residency. Overall, the case was rated relatively highly, with residents scoring the case as 4.3 ± 0.186, 95% confidence interval (1-5 Likert scale, 5 being excellent, n=53) after their assessment was completed. Discussion: Students and residents who participated in the oral board exam formatting found this to be preferable to a traditional lecture and enjoyed the learning environment. Faculty also found this type of participation to be more engaging and were pleased with the ability to perform high-stress assessments with low stakes. The content contained in the case is relevant to all emergency medicine trainees, and this formatting forces the learner to be an active participant in the learning session. The case is a good model for the high-stakes testing of the oral certification exam and is an effective way to test a resident's ability to rapidly assess and manage a life-threatening condition in the ED. Topics: Sickle cell anemia, vaso-occlusive pain crisis, acute chest syndrome, hypoxia, pneumonia, sepsis.

Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.

Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself.

Client binding shifts the populations of dynamic Hsp90 conformations through an allosteric network.

Hsp90 is a molecular chaperone that interacts with a specific set of client proteins and assists their folding. The underlying molecular mechanisms, involving dynamic transitions between open and closed conformations, are still enigmatic. Combining nuclear magnetic resonance, small-angle x-ray scattering, and biochemical experiments, we have identified a key intermediate state of Hsp90 induced by adenosine triphosphate (ATP) binding, in which rotation of the Hsp90 N-terminal domain (NTD) yields a domain arrangement poised for closing. This ATP-stabilized NTD rotation is allosterically communicated across the full Hsp90 dimer, affecting distant client sites. By analyzing the interactions of four distinct clients, i.e., steroid hormone receptors (glucocorticoid receptor and mineralocorticoid receptor), p53, and Tau, we show that client-specific interactions with Hsp90 select and enhance the NTD-rotated state and promote closing of the full-length Hsp90 dimer. The p23 co-chaperone shifts the population of Hsp90 toward the closed state, thereby enhancing client interaction and processing.

Eclampsia.

Audience: Emergency medicine residents and medical students on emergency medicine rotations. Introduction: Eclampsia is an uncommon but important life-threatening obstetrical emergency, complicating 1.5-10 deliveries per 10,000 pregnancies in resource-rich countries.1 If not recognized and treated promptly, there is risk of significant morbidity or death to both mother and baby. Clinically, eclampsia is defined by new-onset seizures or coma in women with preeclampsia.2 Preeclampsia is defined by maternal hypertension after 20 weeks gestation with or without signs of end organ dysfunction, and, like eclampsia, can develop in the postpartum period.1 Eclampsia manifests as new onset generalized tonicclonic seizures. Eclamptic seizures are usually preceded by neurologic symptoms such as severe or atypical headache, visual disturbances, and non-neurologic symptoms such as severe abdominal pain or proteinuria.1 Emergent treatment involves prompt administration of (intravenous) IV magnesium sulfate.2,3,4 Adjuncts include securing the airway if necessary and administration of IV antihypertensive medications. Like preeclampsia, definitive management is by prompt delivery of the fetus if the mother is still pregnant.1 If untreated, maternal mortality is as high as 14%.1 Women who develop eclampsia are at increased risk of obstetric complications in subsequent pregnancies and at higher risk for cardiovascular disease and metabolic disease later in life. Educational Objectives: At the end of this oral boards session, examinees will: 1) Demonstrate ability to obtain a complete medical history including a detailed obstetric history. 2) Demonstrate the ability to perform a detailed physical examination in a postpartum female patient who presents with a seizure. 3) Investigate the broad differential diagnoses which include electrolyte imbalances, brain tumor, meningitis or encephalitis, hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome and eclampsia. 4) List the appropriate laboratory and imaging studies to differentiate eclampsia from other diagnoses (complete blood count, comprehensive metabolic panel, magnesium level, pregnancy testing, urinalysis, and computed tomography [CT] scan of the head). 5) Identify a postpartum eclampsia patient and manage appropriately (administer IV magnesium therapy, administer IV antihypertensive therapy, emergent consultation with an obstetrician). 6) Provide appropriate disposition to the intensive care unit after consulting with an obstetrician. Educational Methods: This was envisioned as an oral board testing case due to the multiple aspects which require emergency care. Residents are expected to assess the seriousness of the patient's condition, elicit critical details from her recent medical history, and synthesize that data in order to treat a medically complex patient. Oral board testing is able to incorporate each of these aspects together and provide the resident with a dynamic learning environment.Oral board testing is a way to assess the resident's ability to rapidly obtain and interpret multiple sources of information simultaneously. By utilizing a case that requires pharmaceutical therapy, the clinical competency committee is able to obtain additional milestones which are sometimes difficult to test in the emergency department itself.Learners were assessed using online evaluation tools available, ie, Google forms. Critical actions were subsequently tied to Emergency Medicine Milestones and the results were compiled and used for resident evaluations and clinical competency. Residents were given verbal feedback immediately after the examination, and they were provided with the scores of their online evaluation after all results were compiled. Research Methods: Learners and instructors provided written feedback after the case was administered to assess for strengths and weaknesses of the case, and modifications were then made to better address concerns. Learners answered written multiple-choice questions on high-level concepts, ie, critical actions, at least one month after this exam was completed. Results: Learners found this a challenging, but enjoyable, way to refresh their knowledge and skills regarding preeclampsia, and this was a highly rated part of their mock oral board examination. Overall, residents rated the session 4.3 (1-5 Likert scale, 5 being Excellent) after the oral board review session was completed. Comments from residents included "haven't seen post-partum preeclampsia in residency" and "challenging to remember magnesium dosing." Discussion: Residents and medical students were evaluated using this method and both enjoyed the activity as a novel way to study as well as exercise their medical knowledge. The content was both highly relevant to the practice of emergency medicine and the format was an effective way to deliver the information to the learners. The case is a good model to evaluate for the high stakes testing of both the written and oral board examinations, but also a way to assess residents' abilities to treat preeclamptic and eclamptic patients in the emergency department. Topics: Eclampsia, preeclampsia, seizures, end-organ damage, hypertensive emergency, altered mental status, neurologic emergency, obstetric emergency.

Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90.

The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

A switch point in the molecular chaperone Hsp90 responding to client interaction.

Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that undergoes large conformational changes during its functional cycle. It has been established that conformational switch points exist in the N-terminal (Hsp90-N) and C-terminal (Hsp90-C) domains of Hsp90, however information for switch points in the large middle-domain (Hsp90-M) is scarce. Here we report on a tryptophan residue in Hsp90-M as a new type of switch point. Our study shows that this conserved tryptophan senses the interaction of Hsp90 with a stringent client protein and transfers this information via a cation-π interaction with a neighboring lysine. Mutations at this position hamper the communication between domains and the ability of a client protein to affect the Hsp90 cycle. The residue thus allows Hsp90 to transmit information on the binding of a client from Hsp90-M to Hsp90-N which is important for progression of the conformational cycle and the efficient processing of client proteins.

Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD.

Genetic and epigenetic alterations in FK506-binding protein 5 ( FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.

Importance of cycle timing for the function of the molecular chaperone Hsp90.

Hsp90 couples ATP hydrolysis to large conformational changes essential for activation of client proteins. The structural transitions involve dimerization of the N-terminal domains and formation of 'closed states' involving the N-terminal and middle domains. Here, we used Hsp90 mutants that modulate ATPase activity and biological function as probes to address the importance of conformational cycling for Hsp90 activity. We found no correlation between the speed of ATP turnover and the in vivo activity of Hsp90: some mutants with almost normal ATPase activity were lethal, and some mutants with lower or undetectable ATPase activity were viable. Our analysis showed that it is crucial for Hsp90 to attain and spend time in certain conformational states: a certain dwell time in open states is required for optimal processing of client proteins, whereas a prolonged population of closed states has negative effects. Thus, the timing of conformational transitions is crucial for Hsp90 function and not cycle speed.