C2-domain abscisic acid-related proteins regulate the dynamics of a plasma membrane H+-ATPase in response to alkali stress.

Arabidopsis (Arabidopsis thaliana) H+-ATPase1 (AHA1), a plasma membrane (PM)-localized H+-ATPase, plays a key role in plant alkali stress tolerance by pumping protons from the cytoplasm to the apoplast. However, its molecular dynamics are poorly understood. We report that many C2-domain ABA-related (CAR) protein family members interact with AHA1 in Arabidopsis. Single or double mutants of CAR1, CAR6, and CAR10 had no obvious phenotype of alkali stress tolerance, while their triple mutants showed significantly higher tolerance to this stress. The disruption of AHA1 largely compromised the increased alkali stress tolerance of the car1car6car10 mutant, revealing a key role of CARs in AHA1 regulation during the plant's response to a high alkali pH. Furthermore, variable angle total internal reflection fluorescence microscopy was used to observe AHA1-mGFP5 in intact Arabidopsis seedlings, revealing the presence of heterogeneous diffusion coefficients and oligomerization states in the AHA1 spots. In the aha1 complementation lines, alkali stress curtailed the residence time of AHA1 at the PM and increased the diffusion coefficient and particle velocity of AHA1. In contrast, the absence of CAR proteins decreased the restriction of the dynamic behavior of AHA1. Our results suggest that CARs play a negative role in plant alkali stress tolerance by interacting with AHA1 and provide a perspective to investigate the regulatory mechanism of PM H+-ATPase activity at the single-particle level.

Recruitment of Ahsa1 to Hsp90 is regulated by a conserved peptide that inhibits ATPase stimulation.

Hsp90 is a molecular chaperone that acts on its clients through an ATP-dependent and conformationally dynamic functional cycle. The cochaperone Accelerator of Hsp90 ATPase, or Ahsa1, is the most potent stimulator of Hsp90 ATPase activity. Ahsa1 stimulates the rate of Hsp90 ATPase activity through a conserved motif, NxNNWHW. Metazoan Ahsa1, but not yeast, possesses an additional 20 amino acid peptide preceding the NxNNWHW motif that we have called the intrinsic chaperone domain (ICD). The ICD of Ahsa1 diminishes Hsp90 ATPase stimulation by interfering with the function of the NxNNWHW motif. Furthermore, the NxNNWHW modulates Hsp90's apparent affinity to Ahsa1 and ATP. Lastly, the ICD controls the regulated recruitment of Hsp90 in cells and its deletion results in the loss of interaction with Hsp90 and the glucocorticoid receptor. This work provides clues to how Ahsa1 conserved regions modulate Hsp90 kinetics and how they may be coupled to client folding status.

CCaP1/CCaP2/CCaP3 interact with plasma membrane H+-ATPases and promote thermo-responsive growth by regulating cell wall modification in Arabidopsis.

Arabidopsis plants adapt to warm temperatures by promoting hypocotyl growth primarily through the basic helix-loop-helix transcription factor PIF4 and its downstream genes involved in auxin responses, which enhance cell division. In the current study, we discovered that cell wall-related calcium-binding protein 2 (CCaP2) and its paralogs CCaP1 and CCaP3 function as positive regulators of thermo-responsive hypocotyl growth by promoting cell elongation in Arabidopsis. Interestingly, mutations in CCaP1/CCaP2/CCaP3 do not affect the expression of PIF4-regulated classic downstream genes. However, they do noticeably reduce the expression of xyloglucan endotransglucosylase/hydrolase genes, which are involved in cell wall modification. We also found that CCaP1/CCaP2/CCaP3 are predominantly localized to the plasma membrane, where they interact with the plasma membrane H+-ATPases AHA1/AHA2. Furthermore, we observed that vanadate-sensitive H+-ATPase activity and cell wall pectin and hemicellulose contents are significantly increased in wild-type plants grown at warm temperatures compared with those grown at normal growth temperatures, but these changes are not evident in the ccap1-1 ccap2-1 ccap3-1 triple mutant. Overall, our findings demonstrate that CCaP1/CCaP2/CCaP3 play an important role in controlling thermo-responsive hypocotyl growth and provide new insights into the alternative pathway regulating hypocotyl growth at warm temperatures through cell wall modification mediated by CCaP1/CCaP2/CCaP3.

A novel lectin receptor kinase gene, AtG-LecRK-I.2, enhances bacterial pathogen resistance through regulation of stomatal immunity in Arabidopsis.

The S-locus lectin receptor kinases (G-LecRKs) have been suggested as receptors for microbe/damage-associated molecular patterns (MAMPs/DAMPs) and to be involved in the pathogen defense responses, but the functions of most G-LecRKs in biotic stress response have not been characterized. Here, we identified a member of this family, G-LecRK-I.2, that positively regulates flg22- and Pseudomonas syringae pv. tomato (Pst) DC3000-induced stomatal closure. G-LecRK-I.2 was rapidly phosphorylated under flg22 treatment and could interact with the FLS2/BAK1 complex. Two T-DNA insertion lines, glecrk-i.2-1 and glecrk-i.2-2, had lower levels of reactive oxygen species (ROS) and nitric oxide (NO) production in guard cells, as compared with the wild-type Col-0, under Pst DC3000 infection. Also, the immunity marker genes CBP60g and PR1 were induced at lower levels under Pst DC3000 hrcC- infection in glecrk-i.2-1 and glecrk-i.2-2. The GUS reporter system also revealed that G-LecRK-I.2 was expressed only in guard cells. We also found that G-LecRK-I.2 could interact H+-ATPase AHA1 to regulate H+-ATPase activity in the guard cells. Taken together, our results show that G-LecRK-I.2 plays an important role in regulating stomatal closure under flg22 and Pst DC3000 treatments and in ROS and NO signaling specifically in guard cells.

Human Aha1's N-terminal extension confers it holdase activity in vitro.

Molecular chaperones are key components of protein quality control system, which plays an essential role in controlling protein homeostasis. Aha1 has been identified as a co-chaperone of Hsp90 known to strongly accelerate Hsp90's ATPase activity. Meanwhile, it is reported that Aha1 could also act as an autonomous chaperone and protect stressed or disordered proteins from aggregation. Here, in this article, a series of in vitro experiments were conducted to verify whether Aha1 has a non-Hsp90-dependent holdase activity and to elucidate the associated molecular mechanism for substrate recognition. According to the results of the refolding assay, the highly conserved N-terminal extension spanning M1 to R16 in Aha1 from higher eukaryotes is responsible for the holdase activity of the protein. As revealed by the NMR data, Aha1's N-terminal extension mainly adopts a disordered conformation in solution and shows no tight contacts with the core structure of Aha1's N-terminal domain. Based on the intrinsically disordered structure feature and the primary sequence of Aha1's N-terminal extension, the fuzzy-type protein-protein interactions involving this specific region and the unfolded substrate proteins are expected. The following mutation analysis data demonstrated that the Van der Waals contacts potentially involving two tryptophans including W4 and W11 do not play a dominant role in the interaction between Aha1 and unfolded maltose binding protein (MBP). Meanwhile, since the high concentration of NaCl could abolish the holdase activity of Aha1, the electrostatic interactions mediated by those charged residues in Aha1's N-terminal extension are thus indicated to play a crucial role in the substrate recognition.

Immunization effect of recombinant Lactobacillus casei displaying Aeromonas veronii Aha1 with an LTB adjuvant in carp.

Aeromonas veronii is an important aquatic zoonotic, which elicits a range of diseases, such as haemorrhagic septicemia. To develop an effective oral vaccine against Aeromonas veronii infection in carp, the Aeromonas veronii adhesion (Aha1) gene was used as a target molecule to attach to intestinal epithelial cells. Two anchored recombinant. Lactic acid bacteria strains (LC-pPG-Aha1 1038 bp and LC-pPG-Aha1-LTB 1383 bp) were constructed by fusing them with the E. coli intolerant enterotoxin B subunit (LTB) gene and using Lactobacillus casei as antigen delivery vector to evaluate immune effects of these in carp. Western blotting and immunofluorescence were used to confirm that protein expression was successful. Additionally, levels of specific IgM in serum and the activities of ACP, AKP, SOD, LYS, C3, C4, and lectin enzymes-were assessed. Cytokines IL-10, IL-1ß, TNF-α, IgZ1, and IgZ2 were measured in the liver, spleen, kidney, intestines, and gills tissue by qRT-PCR, which showed an increasing trend compared with the control group (P < 0.05). A colonization assay showed that the two L. casei recombinants colonized the middle and hind intestines of immunized fish. When immunized carp were experimentally challenged with Aeromonas veronii the relative percentage protection of LC-pPG-Aha1 was 53.57%, and LC-pPG-Aha1-LTB was 60.71%. In conclusion, these results demonstrate that Aha1 is a promising candidate antigen when it is displayed on lactic acid bacteria (Lc-pPG-Aha1 and Lc-pPG-Aha1-LTB) seems promising for a mucosal therapeutic approach. We plan to investigate the molecular mechanism of the L. casei recombinant in regulating the intestinal tissue of carp in future studies.

p23 and Aha1: Distinct Functions Promote Client Maturation.

Hsp90 is a conserved molecular chaperone regulating the folding and activation of a diverse array of several hundreds of client proteins. The function of Hsp90 in client processing is fine-tuned by a cohort of co-chaperones that modulate client activation in a client-specific manner. They affect the Hsp90 ATPase activity and the recruitment of client proteins and can in addition affect chaperoning in an Hsp90-independent way. p23 and Aha1 are central Hsp90 co-chaperones that regulate Hsp90 in opposing ways. While p23 inhibits the Hsp90 ATPase and stabilizes a client-bound Hsp90 state, Aha1 accelerates ATP hydrolysis and competes with client binding to Hsp90. Even though both proteins have been intensively studied for decades, research of the last few years has revealed intriguing new aspects of these co-chaperones that expanded our perception of how they regulate client activation. Here, we review the progress in understanding p23 and Aha1 as promoters of client processing. We highlight the structures of Aha1 and p23, their interaction with Hsp90, and how their association with Hsp90 affects the conformational cycle of Hsp90 in the context of client maturation.

Gene expression and functional analysis of Aha1a and Aha1b in stress response in zebrafish.

Activator of heat shock protein 90 (hsp90) ATPase (Aha1) is a Hsp90 co-chaperone required for Hsp90 ATPase activation. Aha1 is essential for yeast survival and muscle development in C. elegans under elevated temperature and hsp90-deficeiency induced stress conditions. The roles of Aha1 in vertebrates are poorly understood. Here, we characterized the expression and function of Aha1 in zebrafish. We showed that zebrafish genome contains two aha1 genes, aha1a and aha1b, that show distinct patterns of expression during development. Under the normal physiological conditions, aha1a is primarily expressed in skeletal muscle cells of zebrafish embryos, while aha1b is strongly expressed in the head region. aha1a and aha1b expression increased dramatically in response to heat shock induced stress. In addition, Aha1a-GFP fusion protein exhibited a dynamic translocation in muscle cells in response to heat shock. Moreover, upregulation of aha1 expression was also observed in hsp90a1 knockdown embryos that showed a muscle defect. Genetic studies demonstrated that knockout of aha1a, aha1b or both had no detectable effect on embryonic development, survival, and growth in zebrafish. The aha1a and aha1b mutant embryos showed normal muscle development and stress response in response to heat shock. Single or double aha1a and aha1b mutants could grow into normal reproductive adults with normal skeletal muscle structure and morphology compared with wild type control. Together, data from these studies indicate that Aha1a and Aha1b are involved in stress response. However, they are dispensable in zebrafish embryonic development, growth, and survival.

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics.

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and ß-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

Advances towards Understanding the Mechanism of Action of the Hsp90 Complex.

Hsp90 (Heat Shock Protein 90) is an ATP (Adenosine triphosphate) molecular chaperone responsible for the activation and maturation of client proteins. The mechanism by which Hsp90 achieves such activation, involving structurally diverse client proteins, has remained enigmatic. However, recent advances using structural techniques, together with advances in biochemical studies, have not only defined the chaperone cycle but have shed light on its mechanism of action. Hsp90 hydrolysis of ATP by each protomer may not be simultaneous and may be dependent on the specific client protein and co-chaperone complex involved. Surprisingly, Hsp90 appears to remodel client proteins, acting as a means by which the structure of the client protein is modified to allow its subsequent refolding to an active state, in the case of kinases, or by making the client protein competent for hormone binding, as in the case of the GR (glucocorticoid receptor). This review looks at selected examples of client proteins, such as CDK4 (cyclin-dependent kinase 4) and GR, which are activated according to the so-called 'remodelling hypothesis' for their activation. A detailed description of these activation mechanisms is paramount to understanding how Hsp90-associated diseases develop.

Mutations in Hsp90 Cochaperones Result in a Wide Variety of Human Disorders.

The Hsp90 molecular chaperone, along with a set of approximately 50 cochaperones, mediates the folding and activation of hundreds of cellular proteins in an ATP-dependent cycle. Cochaperones differ in how they interact with Hsp90 and their ability to modulate ATPase activity of Hsp90. Cochaperones often compete for the same binding site on Hsp90, and changes in levels of cochaperone expression that occur during neurodegeneration, cancer, or aging may result in altered Hsp90-cochaperone complexes and client activity. This review summarizes information about loss-of-function mutations of individual cochaperones and discusses the overall association of cochaperone alterations with a broad range of diseases. Cochaperone mutations result in ciliary or muscle defects, neurological development or degeneration disorders, and other disorders. In many cases, diseases were linked to defects in established cochaperone-client interactions. A better understanding of the functional consequences of defective cochaperones will provide new insights into their functions and may lead to specialized approaches to modulate Hsp90 functions and treat some of these human disorders.

The disruption of protein-protein interactions with co-chaperones and client substrates as a strategy towards Hsp90 inhibition.

The 90-kiloDalton (kD) heat shock protein (Hsp90) is a ubiquitous, ATP-dependent molecular chaperone whose primary function is to ensure the proper folding of several hundred client protein substrates. Because many of these clients are overexpressed or become mutated during cancer progression, Hsp90 inhibition has been pursued as a potential strategy for cancer as one can target multiple oncoproteins and signaling pathways simultaneously. The first discovered Hsp90 inhibitors, geldanamycin and radicicol, function by competitively binding to Hsp90's N-terminal binding site and inhibiting its ATPase activity. However, most of these N-terminal inhibitors exhibited detrimental activities during clinical evaluation due to induction of the pro-survival heat shock response as well as poor selectivity amongst the four isoforms. Consequently, alternative approaches to Hsp90 inhibition have been pursued and include C-terminal inhibition, isoform-selective inhibition, and the disruption of Hsp90 protein-protein interactions. Since the Hsp90 protein folding cycle requires the assembly of Hsp90 into a large heteroprotein complex, along with various co-chaperones and immunophilins, the development of small molecules that prevent assembly of the complex offers an alternative method of Hsp90 inhibition.

Hsp90 co-chaperones, FKBP52 and Aha1, promote tau pathogenesis in aged wild-type mice.

The microtubule associated protein tau is an intrinsically disordered phosphoprotein that accumulates under pathological conditions leading to formation of neurofibrillary tangles, a hallmark of Alzheimer's disease (AD). The mechanisms that initiate the accumulation of phospho-tau aggregates and filamentous deposits are largely unknown. In the past, our work and others' have shown that molecular chaperones play a crucial role in maintaining protein homeostasis and that imbalance in their levels or activity can drive tau pathogenesis. We have found two co-chaperones of the 90 kDa heat shock protein (Hsp90), FK506-binding protein 52 (FKBP52) and the activator of Hsp90 ATPase homolog 1 (Aha1), promote tau aggregation in vitro and in the brains of tau transgenic mice. Based on this, we hypothesized that increased levels of these chaperones could promote tau misfolding and accumulation in the brains of aged wild-type mice. We tested this hypothesis by overexpressing Aha1, FKBP52, or mCherry (control) proteins in the hippocampus of 9-month-old wild-type mice. After 7 months of expression, mice were evaluated for cognitive and pathological changes. Our results show that FKBP52 overexpression impaired spatial reversal learning, while Aha1 overexpression impaired associative learning in aged wild-type mice. FKBP52 and Aha1 overexpression promoted phosphorylation of distinct AD-relevant tau species. Furthermore, FKBP52 activated gliosis and promoted neuronal loss leading to a reduction in hippocampal volume. Glial activation and phospho-tau accumulation were also detected in areas adjacent to the hippocampus, including the entorhinal cortex, suggesting that after initiation these pathologies can propagate through other brain regions. Overall, our findings suggest a role for chaperone imbalance in the initiation of tau accumulation in the aging brain.

Aha1 Exhibits Distinctive Dynamics Behavior and Chaperone-Like Activity.

Aha1 is the only co-chaperone known to strongly stimulate the ATPase activity of Hsp90. Meanwhile, besides the well-studied co-chaperone function, human Aha1 has also been demonstrated to exhibit chaperoning activity against stress-denatured proteins. To provide structural insights for a better understanding of Aha1's co-chaperone and chaperone-like activities, nuclear magnetic resonance (NMR) techniques were used to reveal the unique structure and internal dynamics features of full-length human Aha1. We then found that, in solution, both the two domains of Aha1 presented distinctive thermal stabilities and dynamics behaviors defined by their primary sequences and three-dimensional structures. The low thermal stability (melting temperature of Aha128-162: 54.45 °C) and the internal dynamics featured with slow motions on the µs-ms time scale were detected for Aha1's N-terminal domain (Aha1N). The aforementioned experimental results suggest that Aha1N is in an energy-unfavorable state, which would therefore thermostatically favor the interaction of Aha1N with its partner proteins such as Hsp90's middle domain. Differently from Aha1N, Aha1C (Aha1's C-terminal domain) exhibited enhanced thermal stability (melting temperature of Aha1204-335: 72.41 °C) and the internal dynamics featured with intermediate motions on the ps-ns time scale. Aha1C's thermal and structural stabilities make it competent for the stabilization of the exposed hydrophobic groove of dimerized Hsp90's N-terminal domain. Of note, according to the NMR data and the thermal shift results, although the very N-terminal region (M1-W27) and the C-terminal relaxin-like factor (RLF) motif showed no tight contacts with the remaining parts of human Aha1, they were identified to play important roles in the recognition of intrinsically disordered pathological α-synuclein.

Human Hsp90 cochaperones: perspectives on tissue-specific expression and identification of cochaperones with similar in vivo functions.

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development.

Controlling protein function by fine-tuning conformational flexibility.

In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein non-specifically. Herein, we directly observe protein regulation under three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational steady states, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We disentangle specific and non-specific effects that control Hsp90's ATPase function, which has remained a puzzle up to now. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results demonstrate how cellular protein regulation works by fine-tuning the conformational state space of proteins.

Proteins play a wide variety of roles in the cell and interact with many other molecules. The behavior of proteins depends on their structure; yet, proteins are often flexible and will change shape, much like a tree in the wind. Nevertheless, for some of the activities that it performs, a protein must adopt one specific shape. Therefore, the likelihood that the protein will take on this specific shape directly determines how efficiently that protein can perform a specific job. The shape of a protein can be regulated by changes at several levels; these could include modifying one of the amino acid building blocks that make up that protein, binding to another protein, or by placing the protein in a part of the cell that is crowded with other large molecules. Schmid and Hugel wanted to understand how these three different types of regulation affect the structure of a protein and how they relate to its activities. The protein Hsp90 was used as a test case. It typically exists with two copies of the protein bound together, either in a parallel or a V-shape. Hsp90 plays several important roles in metabolism and can break down molecules of ATP, the so-called energy currency of the cell. All three types of regulation favored the Hsp90 pairs taking the parallel structure and increased its breakdown of ATP. The results suggest that the Hsp90 pair has a flexible structure, and that reducing this flexibility can improve Hsp90's efficiency in carrying out its role. It was particularly unexpected that the large-scale, unspecific effect of placing the protein in a crowded environment could have such similar results to a small-scale, precise change of a single amino acid within the protein. While all three forms of regulation help to stabilize the parallel structure for Hsp90, they do this through different mechanisms, which influence the speed and the way that the protein transitions between the two structures. Schmid and Hugel believe that these results offer a new perspective on how diversely the shape and function of proteins is controlled at the molecular level, which could have wider implications for medical diagnostics and treatment.

Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone.

Hsp90 plays an important role in health and is a therapeutic target for managing misfolding disease. Compounds that disrupt co-chaperone delivery of clients to Hsp90 target a subset of Hsp90 activities, thereby minimizing the toxicity of pan-Hsp90 inhibitors. Here, we have identified SEW04784 as a first-in-class inhibitor of the Aha1-stimulated Hsp90 ATPase activity without inhibiting basal Hsp90 ATPase. Nuclear magnetic resonance analysis reveals that SEW84 binds to the C-terminal domain of Aha1 to weaken its asymmetric binding to Hsp90. Consistent with this observation, SEW84 blocks Aha1-dependent Hsp90 chaperoning activities, including the in vitro and in vivo refolding of firefly luciferase, and the transcriptional activity of the androgen receptor in cell-based models of prostate cancer and promotes the clearance of phosphorylated tau in cellular and tissue models of neurodegenerative tauopathy. We propose that SEW84 provides a novel lead scaffold for developing therapeutic approaches to treat proteostatic disease.

Aha-type co-chaperones: the alpha or the omega of the Hsp90 ATPase cycle?

Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that plays an essential role in cellular homeostasis. It functions in the context of a structurally dynamic ATP-dependent cycle to promote conformational changes in its clientele to aid stability, maturation, and activation. The client activation cycle is tightly regulated by a cohort of co-chaperone proteins that display specific binding preferences for certain conformations of Hsp90, guiding Hsp90 through its functional ATPase cycle. Aha-type co-chaperones are well-known to robustly stimulate the ATPase activity of Hsp90 but other roles in regulating the functional cycle are being revealed. In this review, we summarize the work done on the Aha-type co-chaperones since the 1990s and highlight recent discoveries with respect to the complexity of Hsp90 cycle regulation.

Co-chaperones TIMP2 and AHA1 Competitively Regulate Extracellular HSP90:Client MMP2 Activity and Matrix Proteolysis.

The extracellular molecular chaperone heat shock protein 90 (eHSP90) stabilizes protease client the matrix metalloproteinase 2 (MMP2), leading to tumor cell invasion. Although co-chaperones are critical modulators of intracellular HSP90:client function, how the eHSP90:MMP2 complex is regulated remains speculative. Here, we report that the tissue inhibitor of metalloproteinases-2 (TIMP2) is a stress-inducible extracellular co-chaperone that binds to eHSP90, increases eHSP90 binding to ATP, and inhibits its ATPase activity. In addition to disrupting the eHSP90:MMP2 complex and terminally inactivating MMP2, TIMP2 loads the client to eHSP90, keeping the protease in a transient inhibitory state. Secreted activating co-chaperone AHA1 displaces TIMP2 from the complex, providing a "reactivating" mechanism for MMP2. Gene knockout or blocking antibodies targeting TIMP2 and AHA1 released by HT1080 cancer cells modify their gelatinolytic activity. Our data suggest that TIMP2 and AHA1 co-chaperones function as a molecular switch that determines the inhibition and reactivation of the eHSP90 client protein MMP2.

Dynamic Aha1 co-chaperone binding to human Hsp90.

Hsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. The co-chaperone Aha1, which is the major ATPase stimulator in eukaryotes, is important for regulation of Hsp90's allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 complex. Here, we characterize the solution structure of unmodified human Hsp90/Aha1 complex using NMR spectroscopy. We show that the 214-kDa complex forms by a two-step binding mechanism and adopts multiple conformations in the absence of nucleotide. Aha1 induces structural changes near Hsp90's nucleotide-binding site, providing a basis for its ATPase-enhancing activity. Our data reveal important aspects of this pivotal chaperone/co-chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.