The structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client maturation state.

The Hsp90 chaperone promotes folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during folding of kinases, nuclear receptors, and tau. Here we determined the cryoelectron microscopy (cryo-EM) structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å, which, together with mutagenesis and crosslinking analyses, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent to Hsp90 client binding sites, whereas a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 is positioned to act on specific client residues presented during Hsp90-catalyzed remodeling.

Amyloid accelerator polyphosphate fits as the mystery density in α-synuclein fibrils.

Aberrant aggregation of α-Synuclein is the pathological hallmark of a set of neurodegenerative diseases termed synucleinopathies. Recent advances in cryo-electron microscopy have led to the structural determination of the first synucleinopathy-derived α-Synuclein fibrils, which contain a non-proteinaceous, "mystery density" at the core of the protofilaments, hypothesized to be highly negatively charged. Guided by previous studies that demonstrated that polyphosphate (polyP), a universally conserved polyanion, significantly accelerates α-Synuclein fibril formation, we conducted blind docking and molecular dynamics simulation experiments to model the polyP binding site in α-Synuclein fibrils. Here, we demonstrate that our models uniformly place polyP into the lysine-rich pocket, which coordinates the mystery density in patient-derived fibrils. Subsequent in vitro studies and experiments in cells revealed that substitution of the 2 critical lysine residues K43 and K45 with alanine residues leads to a loss of all previously reported effects of polyP binding on α-Synuclein, including stimulation of fibril formation, change in filament conformation and stability as well as alleviation of cytotoxicity. In summary, our study demonstrates that polyP fits the unknown electron density present in in vivo α-Synuclein fibrils and suggests that polyP exerts its functions by neutralizing charge repulsion between neighboring lysine residues.

Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.

Structural insights of the p97/VCP AAA+ ATPase: How adapter interactions coordinate diverse cellular functionality.

p97/valosin-containing protein is an essential eukaryotic AAA+ ATPase with diverse functions including protein homeostasis, membrane remodeling, and chromatin regulation. Dysregulation of p97 function causes severe neurodegenerative disease and is associated with cancer, making this protein a significant therapeutic target. p97 extracts polypeptide substrates from macromolecular assemblies by hydrolysis-driven translocation through its central pore. Growing evidence indicates that this activity is highly coordinated by "adapter" partner proteins, of which more than 30 have been identified and are commonly described to facilitate translocation through substrate recruitment or modification. In so doing, these adapters enable critical p97-dependent functions such as extraction of misfolded proteins from the endoplasmic reticulum or mitochondria, and are likely the reason for the extreme functional diversity of p97 relative to other AAA+ translocases. Here, we review the known functions of adapter proteins and highlight recent structural and biochemical advances that have begun to reveal the diverse molecular bases for adapter-mediated regulation of p97 function. These studies suggest that the range of mechanisms by which p97 activity is controlled is vastly underexplored with significant advances possible for understanding p97 regulation by the most known adapters.

Polyphosphate: A Conserved Modifier of Amyloidogenic Processes.

Polyphosphate (polyP), a several billion-year-old biopolymer, is produced in every cell, tissue, and organism studied. Structurally extremely simple, polyP consists of long chains of covalently linked inorganic phosphate groups. We report here the surprising discovery that polyP shows a remarkable efficacy in accelerating amyloid fibril formation. We found that polyP serves as an effective nucleation source for various different amyloid proteins, ranging from bacterial CsgA to human α-synuclein, Aß1-40/42, and Tau. polyP-associated α-synuclein fibrils show distinct differences in seeding behavior, morphology, and fibril stability compared with fibrils formed in the absence of polyP. In vivo, the amyloid-stimulating and fibril-stabilizing effects of polyP have wide-reaching consequences, increasing the rate of biofilm formation in pathogenic bacteria and mitigating amyloid toxicity in differentiated neuroblastoma cells and C. elegans strains that serve as models for human folding diseases. These results suggest that we have discovered a conserved cytoprotective modifier of amyloidogenic processes.

Chemical Features of Polyanions Modulate Tau Aggregation and Conformational States.

The aggregation of tau into insoluble fibrils is a defining feature of neurodegenerative tauopathies. However, tau has a positive overall charge and is highly soluble; so, polyanions, such as heparin, are typically required to promote its aggregation in vitro. There are dozens of polyanions in living systems, and it is not clear which ones might promote this process. Here, we systematically measure the ability of 37 diverse, anionic biomolecules to initiate tau aggregation using either wild-type (WT) tau or the disease-associated P301S mutant. We find that polyanions from many different structural classes can promote fibril formation and that P301S tau is sensitive to a greater number of polyanions (28/37) than WT tau (21/37). We also find that some polyanions preferentially reduce the lag time of the aggregation reactions, while others enhance the elongation rate, suggesting that they act on partially distinct steps. From the resulting structure-activity relationships, the valency of the polyanion seems to be an important chemical feature such that anions with low valency tend to be weaker aggregation inducers, even at the same overall charge. Finally, the identity of the polyanion influences fibril morphology based on electron microscopy and limited proteolysis. These results provide insights into the crucial role of polyanion-tau interactions in modulating tau conformational dynamics with implications for understanding the tau aggregation landscape in a complex cellular environment.

The protein targeting factor Get3 functions as ATP-independent chaperone under oxidative stress conditions.

Exposure of cells to reactive oxygen species (ROS) causes a rapid and significant drop in intracellular ATP levels. This energy depletion negatively affects ATP-dependent chaperone systems, making ROS-mediated protein unfolding and aggregation a potentially very challenging problem. Here we show that Get3, a protein involved in ATP-dependent targeting of tail-anchored (TA) proteins under nonstress conditions, turns into an effective ATP-independent chaperone when oxidized. Activation of Get3's chaperone function, which is a fully reversible process, involves disulfide bond formation, metal release, and its conversion into distinct, higher oligomeric structures. Mutational studies demonstrate that the chaperone activity of Get3 is functionally distinct from and likely mutually exclusive with its targeting function, and responsible for the oxidative stress-sensitive phenotype that has long been noted for yeast cells lacking functional Get3. These results provide convincing evidence that Get3 functions as a redox-regulated chaperone, effectively protecting eukaryotic cells against oxidative protein damage.

Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation.

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis.

The cytochrome P450 enzyme CYP102A1 from Bacillus megaterium is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications. Here, we used size-exclusion chromatography-multiangle light scattering (SEC-MALS) analysis and negative-stain EM to investigate the molecular architecture of CYP102A1. The SEC-MALS analysis yielded a homogeneous peak with an average molecular mass of 235 ± 5 kDa, consistent with homodimeric CYP102A1. The negative-stain EM of dimeric CYP102A1 revealed four distinct lobes, representing the two heme and two reductase domains. Two of the lobes were in close contact, whereas the other two were often observed apart and at the ends of a U-shaped configuration. The overall dimension of the dimer was ∼130 Å. To determine the identity of the lobes, we FLAG-tagged the N or C terminus of CYP102A1 to visualize additional densities in EM and found that anti-FLAG Fab could bind only the N-tagged P450. Single-particle analysis of this anti-Flag Fab-CYP102A1 complex revealed additional density in the N-terminally tagged heme domains, indicating that the heme domains appear flexible, whereas the reductase domains remain tightly associated. The effects of truncation on CYP102A1 dimerization, identification of cross-linked sites by peptide mapping, and molecular modeling results all were consistent with the dimerization of the reductase domain. We conclude that functional CYP102A1 is a compact globular protein dimerized at its reductase domains, with its heme domains exhibiting multiple conformations that likely contribute to the highly efficient catalysis of CYP102A1.

X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition.

Heat shock protein 70 (Hsp70) and Hsp90 are molecular chaperones that play essential roles in tumor growth by stabilizing pro-survival client proteins. However, although the development of Hsp90 inhibitors has benefited from the identification of clients, such as Raf-1 proto-oncogene, Ser/Thr kinase (RAF1), that are particularly dependent on this chaperone, no equivalent clients for Hsp70 have been reported. Using chemical probes and MDA-MB-231 breast cancer cells, we found here that the inhibitors of apoptosis proteins, including c-IAP1 and X-linked inhibitor of apoptosis protein (XIAP), are obligate Hsp70 clients that are rapidly (within ∼3-12 h) lost after inhibition of Hsp70 but not of Hsp90. Mutagenesis and pulldown experiments revealed multiple Hsp70-binding sites on XIAP, suggesting that it is a direct, physical Hsp70 client. Interestingly, this interaction was unusually tight (∼260 nm) for an Hsp70-client interaction and involved non-canonical regions of the chaperone. Finally, we also found that Hsp70 inhibitor treatments caused loss of c-IAP1 and XIAP in multiple cancer cell lines and in tumor xenografts, but not in healthy cells. These results are expected to significantly accelerate Hsp70 drug discovery by providing XIAP as a pharmacodynamic biomarker. More broadly, our findings further suggest that Hsp70 and Hsp90 have partially non-overlapping sets of obligate protein clients in cancer cells.

Client-loading conformation of the Hsp90 molecular chaperone revealed in the cryo-EM structure of the human Hsp90:Hop complex.

Hsp90 is an essential molecular chaperone required for the folding and activation of many hundreds of cellular "client" proteins. The ATP-dependent chaperone cycle involves significant conformational rearrangements of the Hsp90 dimer and interaction with a network of cochaperone proteins. Little is known about the mechanism of client protein binding or how cochaperone interactions modulate Hsp90 conformational states. We have determined the cryo-EM structure of the human Hsp90:Hop complex that receives client proteins from the Hsp70 chaperone. Hop stabilizes an alternate Hsp90 open state, where hydrophobic client-binding surfaces have converged and the N-terminal domains have rotated and match the closed, ATP conformation. Hsp90 is thus simultaneously poised for client loading by Hsp70 and subsequent N-terminal dimerization and ATP hydrolysis. Upon binding of a single Hsp70, the Hsp90:Hop conformation remains essentially unchanged. These results identify distinct functions for the Hop cochaperone, revealing an asymmetric mechanism for Hsp90 regulation and client loading.

Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90ß in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90ß, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90ß in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90ß have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90αα and GR•Hsp90ßß homodimers and no GR•Hsp90αß heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90αß heterodimers. The formation of GR•Hsp90αß heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αß heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM.

Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum.

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.

Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1.

BACKGROUND: The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2, a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission. METHODS: Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. RESULTS: We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of dynamin 2 and stimulates its GTPase activity under physiologic conditions (37°C and 100mM NaCl). At lower ionic strength Arc also stabilizes pre-formed dynamin 2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by dynamin 2. Arc also increases the GTPase activity of dynamin 3, an isoform of implicated in dendrite remodeling, but does not affect the activity of dynamin 1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation. CONCLUSIONS AND GENERAL SIGNIFICANCE: The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity.

Species-dependent ensembles of conserved conformational states define the Hsp90 chaperone ATPase cycle.

The molecular chaperone heat shock protein 90 (Hsp90) is required for the folding and activation of numerous essential signaling proteins. Hsp90 is generally thought to transition between an open (apo) and a closed (ATP) conformation in response to nucleotide. Here, 3D single-particle reconstructions of Escherichia coli and yeast Hsp90 homologs establish the existence of two distinct nucleotide-stabilized conformations (ATP, ADP) in addition to an apo extended state, supporting previous structural work. However, single-particle matching methods reveal that, rather than being irreversibly determined by nucleotide, a species-dependent dynamic conformational equilibrium exists between states. Using crosslinking methods, we trap transient nucleotide-specific states of yeast and human Hsp90 and establish that the apo, ATP, and ADP states are universal. These data support a conserved three-state chaperone cycle where the conformational equilibrium varies between species, implicating evolutionary tuning to meet the particular client protein and metabolic environment of an organism.

Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation.

Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90ß, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/ß. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.

Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.

Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis.

The molecular chaperone Hsp70 activates protein phosphatase 5 (PP5) by binding the tetratricopeptide repeat (TPR) domain.

Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development.

Asymmetric apical domain states of mitochondrial Hsp60 coordinate substrate engagement and chaperonin assembly.

The mitochondrial chaperonin, mitochondrial heat shock protein 60 (mtHsp60), promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin progresses through its ATP-dependent client folding cycle are not clear. Here, we determined cryo-EM structures of a hyperstable disease-associated human mtHsp60 mutant, V72I. Client density is identified in three distinct states, revealing interactions with the mtHsp60 apical domains and C termini that coordinate client positioning in the folding chamber. We further identify an asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60-10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify distinct roles for the apical domains in coordinating client capture and progression through the chaperone cycle, supporting a conserved mechanism of group I chaperonin function.