Structure Of Hsp90 Research Articles

New Brusatol Derivatives as Anti-Settlement Agents Against Barnacles, Targeting HSP90: Design, Synthesis, Biological Evaluation, and Molecular Docking Investigations

The increasing challenge of marine biofouling, mainly due to barnacle settlement, necessitates the development of effective antifoulants with minimal environmental toxicity. In this study, fifteen derivatives of brusatol were synthesized and characterized using 13C-NMR, 1H-NMR, and mass spectrometry. All the semi-synthesized compounds obtained using the Multi-Target-Directed Ligand (MTDL) strategy, when evaluated as anti-settlement agents against barnacles, showed promising activity. Compound 3 exhibited the highest anti-settlement capacity, with an EC50 value of 0.1475 μg/mL, an LC50/EC50 ratio of 42.2922 (>15 indicating low toxicity), and a resuscitation rate of 71.11%, while it showed no significant phenotypic differences in the zebrafish embryos after treatment for 48 h. The toxicity screening of zebrafish also demonstrated the low ecotoxicity of the selected compounds. Furthermore, homology modeling of the HSP90 structure was performed based on related protein sequences in barnacles. Subsequently, molecular docking studies were conducted on HSP90 using these newly synthesized derivatives. Molecular docking analyses showed that most activated derivatives displayed low binding energies with HSP90, aligning well with the biological results. They were found to interact with key residues in the binding site, specifically ARG243, TYR101, and LEU73. These computational findings are anticipated to aid in predicting the enzyme targets of the tested inhibitors and their potential interactions, thus facilitating the design of novel antifoulants in future research endeavors.

Selective Inhibition of hsp90 Paralogs: Uncovering the Role of Helix 1 in Grp94-Selective Ligand Binding.

Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the protein structural elements that drive higher affinity in selective inhibitors. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, PU-H36 derives its higher affinity by utilizing Site 2, a Grp94-specific side pocket adjoining the ATP binding cavity, but in Hsp90 PU-H36 occupies Site 1, a side pocket that is accessible in all paralogs with which it makes lower affinity interactions. The structure of Grp94 in complex with PU-H71 shows only Site 1 binding. While changes in the conformation of helices 4 and 5 in the N-terminal domain occur when ligands bind to Site 1 of both Hsp90 and Grp94, large conformational shifts that also involve helix 1 are associated with the engagement of the Site 2 pocket in Grp94 only. Site 2 in Hsp90 is blocked and its helix 1 conformation is insensitive to ligand binding. To understand the role of helix 1 in ligand selectivity, we tested the binding of PU-H36 and other Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 is the major determinant of selectivity for Site 2 targeted ligands and also influences the rate of ATPase activity in Hsp90 paralogs.

Selective inhibition of hsp90 paralogs: Uncovering the role of helix 1 in Grp94-selective ligand binding.

Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the protein structural elements that drive higher affinity in selective inhibitors. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, PU-H36 derives its higher affinity by utilizing Site 2, a Grp94-specific side pocket adjoining the ATP binding cavity, but in Hsp90 PU-H36 occupies Site 1, a side pocket that is accessible in all paralogs with which it makes lower affinity interactions. The structure of Grp94 in complex with PU-H71 shows only Site 1 binding. While changes in the conformation of helices 4 and 5 in the N-terminal domain occur when ligands bind to Site 1 of both Hsp90 and Grp94, large conformational shifts that also involve helix 1 are associated with the engagement of the Site 2 pocket in Grp94 only. Site 2 in Hsp90 is blocked and its helix 1 conformation is insensitive to ligand binding. To understand the role of helix 1 in ligand selectivity, we tested the binding of PU-H36 and other Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 is the major determinant of selectivity for Site 2 targeted ligands, and also influences the rate of ATPase activity in Hsp90 paralogs.

Structural basis for the dynamic chaperoning of disordered clients by Hsp90.

Molecular chaperone heat shock protein 90 (Hsp90) is a ubiquitous regulator that fine-tunes and remodels diverse client proteins, exerting profound effects on normal biology and diseases. Unraveling the mechanistic details of Hsp90's function requires atomic-level insights into its client interactions throughout the adenosine triphosphate-coupled functional cycle. However, the structural details of the initial encounter complex in the chaperone cycle, wherein Hsp90 adopts an open conformation while engaging with the client, remain elusive. Here, using nuclear magnetic resonance spectroscopy, we determined the solution structure of Hsp90 in its open state, bound to a disordered client. Our findings reveal that Hsp90 uses two distinct binding sites, collaborating synergistically to capture discrete hydrophobic segments within client proteins. This bipartite interaction generates a versatile complex that facilitates rapid conformational sampling. Moreover, our investigations spanning various clients and Hsp90 orthologs demonstrate a pervasive mechanism used by Hsp90 orthologs to accommodate the vast array of client proteins. Collectively, our work contributes to establish a unified conceptual and mechanistic framework, elucidating the intricate interplay between Hsp90 and its clients.

Association of Hsp90 with p53 and Fizzy related homolog (Fzr) synchronizing Anaphase Promoting Complex (APC/C): An unexplored ally towards oncogenic pathway.

The intricate molecular interactions leading to the oncogenic pathway are the consequence of cell cycle modification controlled by a bunch of cell cycle regulatory proteins. The tumor suppressor and cell cycle regulatory proteins work in coordination to maintain a healthy cellular environment. The integrity of this cellular protein pool is perpetuated by heat shock proteins/chaperones, which assist in proper protein folding during normal and cellular stress conditions. Among these versatile groups of chaperone proteins, Hsp90 is one of the significant ATP-dependent chaperones that aid in stabilizing many tumor suppressors and cell cycle regulator protein targets. Recently, studies have revealed that in cancerous cell lines, Hsp90 stabilizes mutant p53, 'the guardian of the genome.' Hsp90 also has a significant impact on Fzr, an essential regulator of the cell cycle having an important role in the developmental process of various organisms, including Drosophila, yeast, Caenorhabditis elegans, and plants. During cell cycle progression, p53 and Fzr coordinately regulate the Anaphase Promoting Complex (APC/C) from metaphase to anaphase transition up to cell cycle exit. APC/C mediates proper centrosome function in the dividing cell. The centrosome acts as the microtubule organizing center for the correct segregation of the sister chromatids to ensure perfect cell division. This review examines the structure of Hsp90 and its co-chaperones, which work in synergy to stabilize proteins such as p53 and Fizzy-related homolog (Fzr) to synchronize the Anaphase Promoting Complex (APC/C). Dysfunction of this process activates the oncogenic pathway leading to the development of cancer. Additionally, an overview of current drugs targeting Hsp90 at various phases of clinical trials has been included.

Despite their structural similarities, the cytosolic isoforms of human Hsp90 show different behaviour in thermal unfolding due to their conformation: An FTIR study

Heat shock proteins 90 (Hsp90) are chaperones that promote the proper folding of other proteins under high temperature stress situations. Hsp90s are highly conserved and ubiquitous proteins, and in mammalian cells, they are localized in the cytoplasm, endoplasmic reticulum, and mitochondria. Cytoplasmic Hsp90 are named Hsp90α and Hsp90β and differ mainly in their expression pattern: Hsp90α is expressed under stress conditions, while Hsp90β is a constitutive protein. Structurally, both share the same characteristics by presenting three well-conserved domains, one of which, the N-terminal domain, has a binding site for ATP to which various drugs targeting this protein, including radicicol, can bind. The protein is mainly found in dimeric form and adopts different conformations depending on the presence of ligands, co-chaperones and client proteins. In this study, some aspects of structure and thermal unfolding of cytoplasmic human Hsp90 were analysed by infrared spectroscopy. The effect on Hsp90β of binding with a non-hydrolysable ATP analogue and radicicol was also examined. The results obtained showed that despite the high similarity in secondary structure the two isoforms exhibit substantial differences in their behaviour during thermal unfolding, as Hsp90α exhibits higher thermal stability, slower denaturation process and different event sequence during unfolding. Ligand binding strongly stabilizes Hsp90β and slightly modifies the secondary structure of the protein as well. Most likely, these structural and thermostability characteristics are closely related to the conformational cycling of the chaperone and its propensity to exist in monomer or dimer form.

An Editorial on the Special Issue ‘Hsp90 Structure, Mechanism and Disease’

Hsp90 is known for its role in the activation of an eclectic set of regulatory and signal transduction proteins [...].

Environmentally relevant concentrations of Triclosan cause transcriptomic and biomolecular alterations in the hatchlings of Labeo rohita

Suppression (p ≤ 0.05) of antioxidative/detoxification (except GPx and CYP3a) and cytoskeletal (except DHPR) genes but induction of metabolic (except for AST and TRY) and heat shock (except HSP60) genes of Labeo rohita hatchlings after 14 days of exposure to environmentally relevant concentrations of Triclosan (0.0063, 0.0126, 0.0252 and 0.06 mg/L) was followed by an increase (p ≤ 0.05) for most of the genes after 10 days recovery period. After recovery, LDH, ALT, CK, CHY, PA, HSP47 and DHPR declined, while SOD, CAT, GST, GR, GPx, CYP1a, CYP3a, AST, AChE, TRY, HSP60, HSP70, HSc71, HSP90 MLP-3, α-tropomyosin, desmin b and lamin b1 increased over exposure. Peak area of biomolecules (except 3290–3296, 2924–2925 and 2852–2855 cm−1) declined (p ≤ 0.01) more after recovery [except for an increase (p ≤ 0.01) at 1398–1401 cm−1]. CYP3a, CK, HSP90, MLP-3 and secondary structure of amide A are the most sensitive markers for the environmentally relevant concentrations of Triclosan.

Structural Changes Induced in Hsp90 by Nitration lead to a Pathological Gain‐of‐function

Heat Shock Protein (Hsp90) is a pro‐survival molecular chaperone essential for the proper folding of a wide range of cellular proteins. It is highly abundant in most cell types and exists as a dimer that interacts with the client proteins. It is well described that the activity of Hsp90 is regulated by post‐translational modifications. We showed that in pathological conditions in which the powerful oxidant peroxynitrite is produced, Hsp90 undergoes nitration at 5 out of 24 tyrosine residues (Y) in its sequence. Further, nitration at Y33 and/or Y56 induces a pathological gain‐of‐function in Hsp90, leading to decreased mitochondrial activity and activation of the purinergic P2X7 receptor, which in motor neurons induces cell death. This is the first nitrated protein with a pathological function described to date. Nitration of Hsp90 is of particular relevance in amyotrophic lateral sclerosis (ALS), a motor neuron degenerative condition with an elusive pathogenesis and scant treatment options. We discovered that Hsp90 is endogenously nitrated in motor neurons in the spinal cord of ALS patients, the cell type that is compromised in this disease process. Establishing the structural changes induced in Hsp90 by nitration that lead to the pathological toxic function is crucial for the development of novel therapeutic strategies for ALS treatment. Here, using a combination of biophysical approaches we show that nitration of Hsp90 at Y33 and Y56, the residues relevant to the toxic function, had a profound impact on Hsp90 structure. To dissect the contribution of nitration at Y33 and/or Y56 on Hsp90 structural changes, we performed analytical ultracentrifugation sedimentation velocity experiments using recombinant Hsp90, peroxynitrite‐treated Hsp90 (fully oxidized protein), and site‐specific nitrated Hsp90 produced by genetic code expansion, carrying nitrotyrosine at either position 33 or 56, or simultaneously at both positions as the sole modification in the protein. Following peroxynitrite treatment, Hsp90 dimer was destabilized, showing a significant increase in the number of monomers, and the formation of oligomeric species. These results were confirmed by negative stain cryo‐electron microscopy, and size exclusion chromatography followed by multiangle light scattering. Site‐specific nitration at Y33 or Y56 was enough to destabilize the dimer conformation, while simultaneous nitration at Y33 and Y56 most closely resembled the structural changes observed after peroxynitrite treatment. In contrast, replacement of Y33 and Y56 by nitration‐resistant phenylalanine decreased dimer destabilization and prevented oligomer formation after peroxynitrite treatment. Together, these results suggest that nitration at Y33 and Y56 is enough to significantly affect the global structure of Hsp90, and that these changes may be responsible for nitrated Hsp90's toxic gain‐of‐function. Thus, targeting these structural changes may lead to the development of drugs that selectively inhibit nitrated Hsp90's pathological activity in ALS, without affecting the function of the unmodified chaperone in normal tissues.

Advances in the role of heat shock protein 90 in prostate cancer.

Prostate cancer is one of the most common tumours in adult men and heat shock proteins play an important biological function in prostate cancer as molecular chaperones involved in the pathogenesis, diagnosis, treatment and prognosis of a wide range of tumours. Among them, increased expression of HSP90, a member of the heat shock protein family, is associated with resistance to prostate cancer denervation and can promote tumour resistance, invasion and bone metastasis, thus making prostate cancer more difficult to treat. Therefore, targeting HSP90 in prostate cancer could be a promising strategy for oncology treatment. This paper reviews the structure and function of HSP90, HSP90-mediated denudation resistance in prostate cancer and HSP90-targeted antitumor therapy, with the aim of providing a new theoretical basis for prostate cancer treatment options in the clinical setting.

Structure of Hsp90-Hsp70-Hop-GR reveals the Hsp90 client-loading mechanism.

Maintaining a healthy proteome is fundamental for the survival of allorganisms1. Integral to this are Hsp90 and Hsp70, molecular chaperones that together facilitate the folding, remodelling and maturation of the many 'client proteins' of Hsp902. The glucocorticoid receptor (GR) is a model client protein that is strictly dependent on Hsp90 and Hsp70 for activity3-7. Chaperoning GR involves a cycle of inactivation by Hsp70; formation of an inactive GR-Hsp90-Hsp70-Hop 'loading' complex; conversion to an active GR-Hsp90-p23 'maturation' complex; and subsequent GR release8. However, to our knowledge,a molecular understanding of this intricate chaperone cycle is lacking for any client protein. Here we report the cryo-electron microscopy structure of the GR-loading complex, in which Hsp70 loads GR onto Hsp90, uncovering the molecular basis of direct coordination by Hsp90 and Hsp70. The structure reveals two Hsp70 proteins, one of which delivers GR and the other scaffolds the Hop cochaperone. Hop interacts with all components of the complex, including GR, and poises Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized through an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR-maturation complexstructure9, we present a complete molecular mechanism of chaperone-dependent client remodelling, and establish general principles of client recognition, inhibition, transfer and activation.

Thermodynamic Dissection of Potency and Selectivity of Cytosolic Hsp90 Inhibitors.

The cytosolic Hsp90-selective inhibitor TAS-116 has an acceptable safety profile and promising antitumor activity in clinical trials. We examined the binding characteristics of TAS-116 and its analogs to determine the impact of the ligand binding mode on selectivity for cytosolic Hsp90. Analyses of the co-crystal structure of Hsp90 and inhibitor TAS-116 suggest that TAS-116 interacts with the ATP-binding pocket, the ATP lid region, and the hydrophobic pocket. A competitive isothermal titration calorimetry analysis confirmed that a small fragment of TAS-116 (THS-510) docks into the lid region and hydrophobic pockets without binding to the ATP-binding pocket. THS-510 exhibited enthalpy-driven binding to Hsp90α and selectively inhibited cytosolic Hsp90 activity. The heat capacity change of THS-510 binding was positive, likely due to the induced conformational rearrangement of Hsp90. Thus, we concluded that interactions with the hydrophobic pocket of Hsp90 determine potency and selectivity of TAS-116 and derivatives for the cytosolic Hsp90 isoform.

Exploring Mechanisms of Communication Switching in the Hsp90-Cdc37 Regulatory Complexes with Client Kinases through Allosteric Coupling of Phosphorylation Sites: Perturbation-Based Modeling and Hierarchical Community Analysis of Residue Interaction Networks.

Understanding molecular principles underlying chaperone-based modulation of kinase client activity is critically important to dissect functions and activation mechanisms of many oncogenic proteins. The recent experimental studies have suggested that phosphorylation sites in the Hsp90 and Cdc37 proteins can serve as conformational communication switches of chaperone regulation and kinase interactions. However, a mechanism of allosteric coupling between phosphorylation sites in the Hsp90 and Cdc37 during client binding is poorly understood, and the molecular signatures underpinning specific roles of phosphorylation sites in the Hsp90 regulation remain unknown. In this work, we employed a combination of evolutionary analysis, coarse-grained molecular simulations together with perturbation-based network modeling and scanning of the unbound and bound Hsp90 and Cdc37 structures to quantify allosteric effects of phosphorylation sites and identify unique signatures that are characteristic for communication switches of kinase-specific client binding. By using network-based metrics of the dynamic intercommunity bridgeness and community centrality, we characterize specific signatures of phosphorylation switches involved in allosteric regulation. Through perturbation-based analysis of the dynamic residue interaction networks, we show that mutations of kinase-specific phosphorylation switches can induce long-range effects and lead to a global rewiring of the allosteric network and signal transmission in the Hsp90-Cdc37-kinase complex. We determine a specific group of phosphorylation sites in the Hsp90 where mutations may have a strong detrimental effect on allosteric interaction network, providing insight into the mechanism of phosphorylation-induced communication switching. The results demonstrate that kinase-specific phosphorylation switches of communications in the Hsp90 may be partly predisposed for their regulatory role based on preexisting allosteric propensities.

Understanding the binding interaction in therapeutic prevention of inflammation by controlling Hsp90 through curcumin and epigallocatechin

Inflammatory checkpoint blockade is the trend of current research targeting Hsp90 with either phytochemical or commercial drugs. It has been extensively studied globally by researchers but nevertheless has been unable to prevent inflammatory disorders successfully. These key considerations set the stage for identification of different combinatorial strategies, curcumin and epigallocatechin to target Hsp90 involved in inflammatory disorders to gain insight into the benefits of this approach. The primary objective in present study is the prior prediction of the binding interactions of curcumin and epigallocatechin with Hsp90 in the living system through computational studies. A suitable animal model sequence for Hsp90 studies was fetched through PHYLOGENY and BLAST. Hsp90 Mus musculus was obtained as a homologous sequence to Hsp90 Homo sapiens and its tertiary structure was homology modeled as its unabridged experimentally derived structure was not available. In additional tertiary model structure was verified using different tools. Single ligand docking and multiple ligand simultaneous docking of curcumin and epigallocatechin to the modeled Hsp90 structure was carried out using the AutoDock tool. In single ligand docking, curcumin had the binding affinity of − 8.3 kcal/mol, while for epigallocatechin − 5.9 kcal/mol and the multiple ligand simultaneous docking binding affinity for Hsp90 to curcumin and epigallocatechin was − 8.4 kcal/mol. Their interface residues were explored for mutational studies and were found to be in an unstabilized state, possibly corresponding to the conformational changes in Hsp90. These strategies emphasize the rationale for combining the modalities, curcumin and epigallocatechin in the successful reduction of Hsp90 expression, henceforth the inflammatory checkpoint would be controlled to augment the anti-inflammatory response.

Single Molecule FRET - A Multi-Environment Ruler for Determining Structure and Dynamics

Studies of multi-domain proteins and multi-protein machines necessitate a deeper understanding of dynamic structure and transient conformations. Therefore, conventional structure determination methods need to be integrated into dynamic detection approaches. We developed a hybrid method that integrates x-ray structure information into self-consistent distance networks based on single-molecule Förster resonance energy transfer (FRET) [1,2]. By analyzing time-correlated distance distributions globally, we can separate real protein dynamics and fluctuations from modelling uncertainties, and ultimately generate time-correlated structural ensembles. On top of that, we reveal correlated small- and large-scale intra-molecular fluctuations from time-resolved, color- and polarization-sensitive fluorescence measurements. We applied our approach to the heat shock protein Hsp90. The chaperone activates large sets of signal transduction proteins often assisted by co-chaperones [3]. These conformation- and nucleotide-dependent processes are not yet comprehensively understood. Our approach resembled the x-ray structure of Hsp90's closed conformation with an RMSD of 2.8 Å. Beyond that, we resolved the previously unknown dynamic open structure of this multidomain protein. The large-scale fluctuations on the lower millisecond timescale might be the basis of a general regulation mechanism. Finally, I want to show how FRET can be orthogonally integrated into cross-disciplinary platforms such as a microfluidics [4] and others, enabling the simultaneous detection of dynamic structure and low-affinity interactions. [1] Hellenkamp B, Wortmann P, Kandzia F, Zacharias M, Hugel T. Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nature Methods 14(2), 174-180 (2017) [2] Hellenkamp B, Schmid S et al. Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study Nature Methods 15, 669-676 (2018) [3] Taipale, M et al. Quantitative Analysis of Hsp90-Client Interactions Reveals Principles of Substrate Recognition. Cell 140(5), 987-1001 (2012) [4] Hellenkamp B, Thurn J, Stadlmeier M, Hugel T., submitted

Hsp90 Structure and Function in Cancer

: Heat shock protein 90 (HSP90) serves an essential role in stability and function of over-expressed proteins that promote malignancy. HSP90 guides various cellular and physiological processes/functions such as cell growth, cell survival and differentiation, hormone signaling, trafficking, response to cellular stress, and apoptosis. Conversely, HSP90 during oncogenesis causes malignant transformation and is critical for the maintenance and maturation of a broad range of mutated proteins activated and/or over-expressed in signaling pathways, promoting cancer cell growth and/or survival. Therefore, HSP90 is an attractive target strategy for tumor treatment. We described HSP90 structure and function in normal cells and in malignancy.

Structure and pro-toxic mechanism of the human Hsp90/PPIase/Tau complex

The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer’s disease-related protein Tau. We reveal that the FKBP51/Hsp90 complex, which synergizes to promote toxic Tau oligomers in vivo, is highly dynamic and stabilizes the extended conformation of the Hsp90 dimer resulting in decreased Hsp90 ATPase activity. Within the ternary Hsp90/FKBP51/Tau complex, Hsp90 serves as a scaffold that traps the PPIase and nucleates multiple conformations of Tau’s proline-rich region next to the PPIase catalytic pocket in a phosphorylation-dependent manner. Our study defines a conceptual model for dynamic Hsp90/co-chaperone/client recognition.

Dissecting Structure-Encoded Determinants of Allosteric Cross-Talk between Post-Translational Modification Sites in the Hsp90 Chaperones

Post-translational modifications (PTMs) represent an important regulatory instrument that modulates structure, dynamics and function of proteins. The large number of PTM sites in the Hsp90 proteins that are scattered throughout different domains indicated that synchronization of multiple PTMs through a combinatorial code can be invoked as an important mechanism to orchestrate diverse chaperone functions and recognize multiple client proteins. In this study, we have combined structural and coevolutionary analysis with molecular simulations and perturbation response scanning analysis of the Hsp90 structures to characterize functional role of PTM sites in allosteric regulation. The results reveal a small group of conserved PTMs that act as global mediators of collective dynamics and allosteric communications in the Hsp90 structures, while the majority of flexible PTM sites serve as sensors and carriers of the allosteric structural changes. This study provides a comprehensive structural, dynamic and network analysis of PTM sites across Hsp90 proteins, identifying specific role of regulatory PTM hotspots in the allosteric mechanism of the Hsp90 cycle. We argue that plasticity of a combinatorial PTM code in the Hsp90 may be enacted through allosteric coupling between effector and sensor PTM residues, which would allow for timely response to structural requirements of multiple modified enzymes.

Structural Insights into the Regulation Mechanism of HSP90 by Co-chaperone AHA1

Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of “client” proteins, especially those involved in signaling and regulatory processes. This Hsp90 chaperone machinery is also essential for maintaining cellular protein homeostasis and for dealing with proteotoxic stress. The client maturation cycle is regulated by a large number of co-chaperones that sequentially interact with Hsp90 throughout its ATPase cycle. One of the most important, but poorly understood co-chaperones in eukaryotes is Activator of Hsp90 ATPase homolog 1 (Aha1). As a potent stimulator of the Hsp90 ATPase activity, Aha1 is thought to accelerate client protein remodeling. A knockout of Aha1 in yeast severely compromises the maturation of different Hsp90 client proteins. However, Aha1 overexpression inhibits the rate of Hsp90-dependent refolding of denatured luciferase, as well as cystic fibrosis associated protein CFTR. How Aha1, as an Hsp90 activator, facilitates ATP hydrolysis while inhibiting client maturation remains enigmatic. To better understand this question, we have determined the structure of the full-length 200-kDa Hsp90/Aha1 complex at 3.8 Å resolution using cryo-electron microscopy. When bound to Aha1, the Hsp90 was captured in the closed conformation, similar to the X-ray crystal structure of Hsp90 bound with inhibitory co-chaperone p23. However, the binding interface is completely different from that used by p23 as well as that observed by crystallography of a fragment of Aha bound to a fragment of Hsp90. In our structure, the C-terminal domain of Aha1 stabilizes the Hsp90 closed dimer by bridging the C-terminal domain of one protomer and the middle domain/N-terminal domain of the other protomer. This structure begins to explain the Aha1 inhibitory effect on Hsp90 and also provides a basis for designing co-chaperone specific inhibitors to regulate Hsp90 related diseases.

The Structural Asymmetry of Mitochondrial Hsp90 (Trap1) Determines Fine Tuning of Functional Dynamics.

The Hsp90 family of molecular chaperones oversees the folding of a wide range of client proteins. Hsp90 is a homodimer, whose conformational states and functions are regulated by ATP-binding and hydrolysis. The crystal structure of mitochondrial Hsp90 (Trap1) showed that one of the two protomers in the active state is buckled, resulting in an asymmetric conformation. The asymmetry between the two protomers corresponds to the broadly conserved region responsible for client binding. Moreover, asymmetry determines differential hydrolysis for each protomer, with the buckled conformation favoring ATP processing. Experimental results show that after the first hydrolysis the dimer flips to a different asymmetric state while remaining in a closed conformation for the second hydrolysis. In this model, asymmetry plays a key role in the mechanism that drives chaperone function. Herein, we investigate the nucleotide-dependent internal dynamics of Trap1 with computational approaches. Our results shed light on the relationship between the nucleotide state in the N-terminal domain and the asymmetric modulation of the dynamic and structural properties of the client-binding region in the Middle domain. According to our analysis, this is the region that undergoes the most intense dynamic modulation upon nucleotide exchange. This result provides molecular insights into the roles of structural asymmetry in the regulation of Trap1 and suggests that this substructure is a promising target to modulate the functionally oriented aspects of Trap1 dynamics, therefore opening fresh opportunities for the design of selective therapeutics for Trap1-dependent diseases.