X-ray quasi-periodic eruptions from two previously quiescent galaxies.

Quasi-periodic eruptions (QPEs) are very-high-amplitude bursts of X-ray radiation recurring every few hours and originating near the central supermassive black holes of galactic nuclei1,2. It is currently unknown what triggers these events, how long they last and how they are connected to the physical properties of the inner accretion flows. Previously, only two such sources were known, found either serendipitously or in archival data1,2, with emission lines in their optical spectra classifying their nuclei as hosting an actively accreting supermassive black hole3,4. Here we report observations of QPEs in two further galaxies, obtained with a blind and systematic search of half of the X-ray sky. The optical spectra of these galaxies show no signature of black hole activity, indicating that a pre-existing accretion flow that is typical of active galactic nuclei is not required to trigger these events. Indeed, the periods, amplitudes and profiles of the QPEs reported here are inconsistent with current models that invoke radiation-pressure-driven instabilities in the accretion disk5-9. Instead, QPEs might be driven by an orbiting compact object. Furthermore, their observed properties require the mass of the secondary object to be much smaller than that of the main body10, and future X-ray observations may constrain possible changes in their period owing to orbital evolution. This model could make QPEs a viable candidate for the electromagnetic counterparts of so-called extreme-mass-ratio inspirals11-13, with considerable implications for multi-messenger astrophysics and cosmology14,15.

Dynamics of heat shock protein 90 C-terminal dimerization is an important part of its conformational cycle.

The molecular chaperone heat shock protein 90 (Hsp90) is an important and abundant protein in eukaryotic cells, essential for the activation of a large set of signal transduction and regulatory proteins. During the functional cycle, the Hsp90 dimer performs large conformational rearrangements. The transient N-terminal dimerization of Hsp90 has been extensively investigated, under the assumption that the C-terminal interface is stably dimerized. Using a fluorescence-based single molecule assay and Hsp90 dimers caged in lipid vesicles, we were able to separately observe and kinetically analyze N- and C-terminal dimerizations. Surprisingly, the C-terminal dimer opens and closes with fast kinetics. The occupancy of the unexpected C-terminal open conformation can be modulated by nucleotides bound to the N-terminal domain and by N-terminal deletion mutations, clearly showing a communication between the two terminal domains. Moreover our findings suggest that the C- and N-terminal dimerizations are anticorrelated. This changes our view on the conformational cycle of Hsp90 and shows the interaction of two dimerization domains.

Protein aggregation as a cause for disease.

The ability of proteins to fold into a defined and functional conformation is one of the most fundamental processes in biology. Certain conditions, however, initiate misfolding or unfolding of proteins. This leads to the loss of functional protein or it can result in a wide range of diseases. One group of diseases, which includes Alzheimer's, Parkinson's, Huntington's disease, and the transmissible spongiform encephalopathies (prion diseases), involves deposition of aggregated proteins. Normally, such protein aggregates are not found in properly functioning biological systems, because a variety of mechanisms inhibit their formation. Understanding the nature of these protective mechanisms together with the understanding of factors reducing or deactivating the natural protection machinery will be crucial for developing strategies to prevent and treat these disastrous diseases.

Hsp70 and Hsp90--a relay team for protein folding.

Molecular chaperones are a functionally defined set of proteins which assist the structure formation of proteins in vivo. Without certain protective mechanisms, such as binding nascent polypeptide chains by molecular chaperones, cellular protein concentrations would lead to misfolding and aggregation. In the mammalian system, the molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins, like steroid hormone receptors, transcription factors, and kinases, some of which are involved in cancer progression. Hsp70 and Hsp90 form a multichaperone complex, in which both are connected by a third protein called Hop. The connection of and the interplay between the two chaperone machineries is of crucial importance for cell viability. This review provides a detailed view of the Hsp70 and Hsp90 machineries, their cofactors and their mode of regulation. It summarizes the current knowledge in the field, including the ATP-dependent regulation of the Hsp70/Hsp90 multichaperone cycle and elucidates the complex interplay and their synergistic interaction.

Review: a structural view of the GroE chaperone cycle.

The GroE chaperone system consists of two ring-shaped oligomeric components whose association creates different functional states. The most remarkable property of the GroE system is the ability to fold proteins under conditions where spontaneous folding cannot occur. To achieve this, a fully functional system consisting of GroEL, the cochaperone GroES, and ATP is necessary. Driven by ATP binding and hydrolysis, this system cycles through different conformational stages, which allow binding, folding, and release of substrate proteins. Some aspects of the ATP-driven reaction cycle are still under debate. One of these open questions is the importance of so-called "football" complexes consisting of GroEL and two bound GroES rings. Here, we summarize the evidence for the functional relevance of these complexes and their involvement in the efficient folding of substrate proteins.

Localization of the chaperone domain of FKBP52.

FKBP52, a multidomain peptidyl prolyl cis/trans-isomerase (PPIase), is found in complex with the chaperone Hsp90 and the co-chaperone p23. It displays both PPIase and chaperone activity in vitro. To localize these two activities to specific regions of the protein, we created and analyzed a set of fragments of FKBP52. The PPIase activity toward both peptides and proteins is confined entirely to domain 1 (amino acids 1-148). The chaperone activity, however, resides in the C-terminal part of FKBP52, mainly in the region between amino acids 264 and 400 (domain 3). Interestingly, this domain also contains the tetratricopeptide repeats, which are responsible for the binding to C-terminal amino acids of Hsp90. Competition assays with a C-terminal Hsp90 peptide suggest that the non-native protein and Hsp90 are bound by different regions within this domain.

Hsp90: chaperoning signal transduction.

Hsp90 is an ATP dependent molecular chaperone involved in the folding and activation of an unknown number of substrate proteins. These substrate proteins include protein kinases and transcription factors. Consistent with this task, Hsp90 is an essential protein in all eucaryotes. The interaction of Hsp90 with its substrate proteins involves the transient formation of multiprotein complexes with a set of highly conserved partner proteins. The specific function of each component in the processing of substrates is still unknown. Large ATP-dependent conformational changes of Hsp90 occur during the hydrolysis reaction and these changes are thought to drive the chaperone cycle. Natural inhibitors of the ATPase activity, like geldanamycin and radicicol, block the processing of Hsp90 substrate proteins. As many of these substrates are critical elements in signal transduction, Hsp90 seems to introduce an additional level of regulation.

Coordinated ATP hydrolysis by the Hsp90 dimer.

The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.

The alternatively folded state of the antibody C(H)3 domain.

The C(H)3 domain of antibodies is characterized by two antiparallel beta-sheets forming a disulfide-linked sandwich-like structure. At acidic pH values and low ionic strength, C(H)3 becomes completely unfolded. The addition of salt transforms the acid-unfolded protein into an alternatively folded state exhibiting a characteristic secondary structure. The transition from native to alternatively folded C(H)3 is a fast reaction. Interestingly, this reaction involves the formation of a defined oligomer consisting of 12-14 subunits. Association is completely reversible and the native dimer is quantitatively reformed at neutral pH. This alternatively folded protein is remarkably stable against thermal and chemical denaturation and the unfolding transitions are highly cooperative. With a t(m) of 80 degrees C, the stability of the alternatively folded state is comparable to that of the native state of C(H)3. The defined oligomeric structure of C(H)3 at pH 2 seems to be a prerequisite for the cooperative unfolding transitions.

Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p.

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Chem. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.

Functional analysis of the Hsp90-associated human peptidyl prolyl cis/trans isomerases FKBP51, FKBP52 and Cyp40.

Large peptidyl-prolyl cis/trans isomerases (PPIases) are important components of the Hsp90 chaperone complex. In mammalian cells, either Cyp40, FKBP51 or FKBP52 is incorporated into these complexes. It has been suggested that members of this protein family exhibit both prolyl isomerase and chaperone activity. Here we define the structural and functional properties of the three mammalian large PPIases. We find that in all cases two PPIase monomers bind to an Hsp90 dimer. However, the affinities of the PPIases are different with FKBP52 exhibiting the strongest interaction and Cyp40 the weakest. Furthermore, in the mammalian system, in contrast to the yeast system, the catalytic activity of prolyl isomerization corresponds well to that of the respective small PPIases. Interestingly, Cyp40 and FKBP51 are the more potent chaperones. Thus, it seems that both the affinity for Hsp90 and the differences in their chaperone properties, which may reflect their interaction with the non-native protein in the Hsp90 complex, are critical for the selective incorporation of a specific large PPIase.

Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv.

Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.

The crystal structure of the fab fragment of the monoclonal antibody MAK33. Implications for folding and interaction with the chaperone bip.

The Fab fragment of the murine monoclonal antibody, MAK33, directed against human creatine kinase of the muscle-type, was crystallized and the three-dimensional structure was determined to 2.9 A. The antigen-binding surface of MAK33 shows a convex overall shape typical for immunoglobulins binding large antigens. The structure allows us to analyze the environment of cis-prolyl-peptide bonds whose isomerization is of key importance in the folding process. These residues seem to be involved with not only domain stability but also seem to play a role in the association of heavy and light chains, reinforcing the importance of beta-strand recognition in antibody assembly. The structure also allows the localization of segments of primary sequence postulated to represent binding sites for the ER-specific chaperone BiP within the context of the entire Fab fragment. These sequences are found primarily in beta-strands that are necessary for interactions between the individual domains.

C-terminal regions of Hsp90 are important for trapping the nucleotide during the ATPase cycle.

Hsp90 is an abundant molecular chaperone that functions in an ATP-dependent manner in vivo. The ATP-binding site is located in the N-terminal domain of Hsp90. Here, we dissect the ATPase cycle of Hsp90 kinetically. We find that Hsp90 binds ATP with a two-step mechanism. The rate-limiting step of the ATPase cycle is the hydrolysis of ATP. Importantly, ATP becomes trapped and committed to hydrolyze during the cycle. In the isolated ATP-binding domain of Hsp90, however, the bound ATP was not committed and the turnover numbers were markedly reduced. Analysis of a series of truncation mutants of Hsp90 showed that C-terminal regions far apart in sequence from the ATP-binding domain are essential for trapping the bound ATP and for maximum hydrolysis rates. Our results suggest that ATP binding and hydrolysis drive conformational changes that involve the entire molecule and lead to repositioning of the N and C-terminal domains of Hsp90.

Cpr6 and Cpr7, two closely related Hsp90-associated immunophilins from Saccharomyces cerevisiae, differ in their functional properties.

Hsp90 is an abundant cytosolic molecular chaperone. It controls the folding of target proteins including steroid hormone receptors and kinases in complex with several partner proteins. Prominent members of this protein family are large peptidyl prolyl cis/trans isomerases (PPIases), which catalyze the cis/trans isomerization of prolyl peptide bonds in proteins and possess chaperone activity. In Saccharomyces cerevisiae, two closely related large Hsp90-associated PPIases, Cpr6 and Cpr7, exist. We show here that these homologous proteins bind with comparable affinity to Hsp90 but exhibit significant structural and functional differences. Cpr6 is more stable than Cpr7 against thermal denaturation and displays an up to 100-fold higher PPIase activity. In contrast, the chaperone activity of Cpr6 is much lower than that of Cpr7. Based on these results we suggest that the two immunophilins perform overlapping but not identical tasks in the Hsp90 chaperone cycle.

BiP and PDI cooperate in the oxidative folding of antibodies in vitro.

Immunoglobulin heavy chain binding protein (BiP), a member of the Hsp70 chaperone family, and the oxidoreductase protein-disulfide isomerase (PDI) play an important role in the folding and oxidation of proteins in the endoplasmic reticulum. However, it was not clear whether both cooperate in this process. We show here that BiP and PDI act synergistically in the in vitro folding of the denatured and reduced Fab fragment. Several ATP-dependent cycles of binding, release, and rebinding of the unfolded antibody chains by BiP are required for efficient reactivation. Our data suggest that in the absence of BiP unfolded antibody chains collapse rapidly upon refolding, rendering cysteine side chains inaccessible for PDI. BiP binds the unfolded polypeptide chains and keeps them in a conformation in which the cysteine residues are accessible for PDI. These findings support the idea of a network of folding helper proteins in the endoplasmic reticulum, which makes this organelle a dedicated protein-processing compartment.

Limits of protein folding inside GroE complexes.

The GroE chaperones of Escherichia coli promote the folding of other proteins under conditions where no spontaneous folding occurs. One requirement for this reaction is the trapping of the nonnative protein inside the chaperone complex. Encapsulation may be important to prevent unfavorable intermolecular interactions during folding. We show here that, especially for oligomeric proteins, the timing of encapsulation and release is of critical importance. If this cycle is decelerated, misfolding is observed inside functional chaperone complexes.

Mouse Hsp25, a small shock protein. The role of its C-terminal extension in oligomerization and chaperone action.

Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short, polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild-type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize alpha-lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature. 1H NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra C-terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced alpha-lactalbumin by 1H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian sHsps is required for full chaperone activity.