Catalytic Relaxation of Kinetically Trapped Intermediates by DNA Chaperones.

Common in biomacromolecules, kinetically trapped misfolded intermediates are often detrimental to the structures, properties, or functions of proteins or nucleic acids. Nature employs chaperone proteins but not nucleic acids to escort intermediates to correct conformations. Herein, we constructed a Jablonski-like diagram of a mechanochemical cycle in which individual DNA hairpins were mechanically unfolded to high-energy states, misfolded into kinetically trapped states, and catalytically relaxed back to ground-state hairpins by a DNA chaperone. The capacity of catalytic relaxation was demonstrated in a 1D DNA hairpin array mimicking nanoassembled materials. At ≥1 µM, the diffusive (or self-walking) DNA chaperone converted the entire array of misfolded intermediates to correct conformation in less than 15 s, which is essential to rapidly prepare homogeneous nanoassemblies. Such an efficient self-walking amplification increases the signal-to-noise ratio, facilitating catalytic relaxation to recognize a 1 fM DNA chaperone in 10 min, a detection limit comparable to the best biosensing strategies.

Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes.

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii.

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

Nucleotide-induced ClpC oligomerization and its non-preferential association with ClpP isoforms of pathogenic Leptospira.

Bacterial caseinolytic protease-chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class-I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide-induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non-preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade ß-casein or FITC-casein in an energy-independent manner. Adding either ATP or ATPγS further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of hetero-tetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

Dual mode of DDX3X as an ATP-dependent RNA helicase and ATP-independent nucleic acid chaperone.

Human DDX3X, an important member of the DEAD-box family RNA helicases, plays a crucial role in RNA metabolism and is involved in cancer development, viral infection, and neurodegenerative disease. Although there have been many studies on the physiological functions of human DDX3X, issues regarding its exact targets and mechanisms of action remain unclear. In this study, we systematically characterized the biochemical activities and substrate specificity of DDX3X. The results demonstrate that DDX3X is a bidirectional RNA helicase to unwind RNA duplex and RNA-DNA hybrid driven by ATP. DDX3X also has nucleic acid annealing activity, especially for DNA. More importantly, it can function as a typical nucleic acid chaperone which destabilizes highly structured DNA and RNA in an ATP-independent manner and promotes their annealing to form a more stable structure. Further truncation mutations confirmed that the highly disordered N-tail and C-tail are critical for the biochemical activities of DDX3X. They are functionally complementary, with the N-tail being crucial. These results will shed new light on our understanding of the molecular mechanism of DDX3X in RNA metabolism and DNA repair, and have potential significance for the development of antiviral/anticancer drugs targeting DDX3X.

Chaperone-mediated MHC-I peptide exchange in antigen presentation.

This work focuses on molecules that are encoded by the major histocompatibility complex (MHC) and that bind self-, foreign- or tumor-derived peptides and display these at the cell surface for recognition by receptors on T lymphocytes (T cell receptors, TCR) and natural killer (NK) cells. The past few decades have accumulated a vast knowledge base of the structures of MHC molecules and the complexes of MHC/TCR with specificity for many different peptides. In recent years, the structures of MHC-I molecules complexed with chaperones that assist in peptide loading have been revealed by X-ray crystallography and cryogenic electron microscopy. These structures have been further studied using mutagenesis, molecular dynamics and NMR approaches. This review summarizes the current structures and dynamic principles that govern peptide exchange as these relate to the process of antigen presentation.

HtpG-A Major Virulence Factor and a Promising Vaccine Antigen against Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading global cause of death f rom an infectious bacterial agent. Therefore, limiting its epidemic spread is a pressing global health priority. The chaperone-like protein HtpG of M. tuberculosis (Mtb) is a large dimeric and multi-domain protein with a key role in Mtb pathogenesis and promising antigenic properties. This dual role, likely associated with the ability of Heat Shock proteins to act both intra- and extra-cellularly, makes HtpG highly exploitable both for drug and vaccine development. This review aims to gather the latest updates in HtpG structure and biological function, with HtpG operating in conjunction with a large number of chaperone molecules of Mtb. Altogether, these molecules help Mtb recovery after exposure to host-like stress by assisting the whole path of protein folding rescue, from the solubilisation of aggregated proteins to their refolding. Also, we highlight the role of structural biology in the development of safer and more effective subunit antigens. The larger availability of structural information on Mtb antigens and a better understanding of the host immune response to TB infection will aid the acceleration of TB vaccine development.

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity.

HdeA and HdeB are dimeric ATP-independent acid-stress chaperones, which protect the periplasmic proteins of enteric bacteria at pH 2.0 and 4.0, respectively, during their passage through the acidic environment of the mammalian stomach. Despite being structurally similar, they exhibit distinct functional pH optima and conformational prerequisite for their chaperone action. HdeA undergoes a dimer-to-monomer transition at pH 2.0, whereas HdeB remains dimeric at pH 4.0. The monomerization of HdeA exposes its hydrophobic motifs, which facilitates its interaction with the partially folded substrates. How HdeB functions despite maintaining its dimeric conformation has been poorly elucidated in the literature. Herein, we characterized the conformational states and stability of HdeB at its physiologically relevant pH and compared the data with those of HdeA. At pH 4.0, HdeB exhibited distinct spectroscopic signatures and higher stability against heat and guanidine-HCl-induced denaturation than at pH 7.5. We affirm that the pH 4.0 conformer of HdeB was distinct from that at pH 7.5 and that these two conformational states were hierarchically unrelated. Salt-bridge mutations that perturbed HdeB's intersubunit interactions resulted in the loss of its stability and function at pH 4.0. In contrast, mutations affecting intrasubunit interactions enhanced its function, albeit with a reduction in stability. These findings suggest that, unlike HdeA, HdeB acts as a noncanonical chaperone, where pH-dependent stability and conformational rearrangement at pH 4.0 play a core role in its chaperone function rather than its surface hydrophobicity. Such rearrangement establishes a stability-function trade-off that allows HdeB to function while maintaining its stable dimeric state.

Structure of the human 20S U5 snRNP.

The 20S U5 small nuclear ribonucleoprotein particle (snRNP) is a 17-subunit RNA-protein complex and a precursor of the U4/U6.U5 tri-snRNP, the major building block of the precatalytic spliceosome. CD2BP2 is a hallmark protein of the 20S U5 snRNP, absent from the mature tri-snRNP. Here we report a high-resolution cryogenic electron microscopy structure of the 20S U5 snRNP, shedding light on the mutually exclusive interfaces utilized during tri-snRNP assembly and the role of the CD2BP2 in facilitating this process.

Navigating the complexities of multi-domain protein folding.

Proteome complexity has expanded tremendously over evolutionary time, enabling biological diversification. Much of this complexity is achieved by combining a limited set of structural units into long polypeptides. This widely used evolutionary strategy poses challenges for folding of the resulting multi-domain proteins. As a consequence, their folding differs from that of small single-domain proteins, which generally fold quickly and reversibly. Co-translational processes and chaperone interactions are important aspects of multi-domain protein folding. In this review, we discuss some of the recent experimental progress toward understanding these processes.

Structural basis of human U5 snRNP late biogenesis and recycling.

Pre-mRNA splicing by the spliceosome requires the biogenesis and recycling of its small nuclear ribonucleoprotein (snRNP) complexes, which are consumed in each round of splicing. The human U5 snRNP is the ~1 MDa 'heart' of the spliceosome and is recycled through an unknown mechanism involving major architectural rearrangements and the dedicated chaperones CD2BP2 and TSSC4. Late steps in U5 snRNP biogenesis similarly involve these chaperones. Here we report cryo-electron microscopy structures of four human U5 snRNP-CD2BP2-TSSC4 complexes, revealing how a series of molecular events primes the U5 snRNP to generate the ~2 MDa U4/U6.U5 tri-snRNP, the largest building block of the spliceosome.

Polymorphic Self-Assembly with Procedural Flexibility for Monodisperse Quaternary Protein Structures of DegQ Enzymes.

As large molecular tertiary structures, some proteins can act as small robots that find, bind, and chaperone target protein clients, showing the potential to serve as smart building blocks in self-assembly fields. Instead of using such intrinsic functions, most self-assembly methodologies for proteins aim for de novo-designed structures with accurate geometric assemblies, which can limit procedural flexibility. Here, a strategy enabling polymorphic clustering of quaternary proteins, exhibiting simplicity and flexibility of self-assembling paths for proteins in forming monodisperse quaternary cage particles is presented. It is proposed that the enzyme protomer DegQ, previously solved at low resolution, may potentially be usable as a threefold symmetric building block, which can form polyhedral cages incorporated by the chaperone action of DegQ in the presence of protein clients. To obtain highly monodisperse cage particles, soft, and hence, less resistive client proteins, which can program the inherent chaperone activity of DegQ to efficient formations of polymorphic cages, depending on the size of clients are utilized. By reconstructing the atomic resolution cryogenic electron microscopy DegQ structures using obtained 12- and 24-meric clusters, the polymorphic clustering of DegQ enzymes is validated in terms of soft and rigid domains, which will provide effective routes for protein self-assemblies with procedural flexibility.

Exploiting cyclodextrins as artificial chaperones to enhance enzyme protection through supramolecular engineering.

We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of ß-cyclodextrin (ß-CD) covalently embedded in an ultrathin organosilica layer. Putative interaction points of this artificial chaperone system with the surface of the selected enzyme were studied in silico using a protein energy landscape exploration simulation algorithm. We show that this enzyme shielding method allows for drastic enhancement of enzyme stability under thermal and chemical stress conditions, along with broadening the optimal temperature range of the biocatalyst. The presence of the ß-CD macrocycle within the protective layer supports protein refolding after treatment with a surfactant.

Intrinsically disordered Pseudomonas chaperone FapA slows down the fibrillation of major biofilm-forming functional amyloid FapC.

Functional bacterial amyloids play a crucial role in the formation of biofilms, which mediate chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC amyloid fibrillar protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution nuclear magnetic resonance (NMR), we recently showed that both FapC and FapA are intrinsically disordered proteins (IDPs). Here, the secondary structure propensities (SSPs) are compared to alphafold (DeepMind, protein structure prediction tool/algorithm: https://alphafold.ebi.ac.uk/) models. We further demonstrate that the FapA chaperone interacts with FapC and significantly slows down the formation of FapC fibrils. Our NMR titrations reveal ~ 18% of the resonances show FapA-induced chemical shift perturbations (CSPs), which has not been previously observed, the largest being for A82, N201, C237, C240, A241, and G245. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the repeat-1 (R1)/loop-2 (L2) and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, ~ 90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC, the largest being for S63, A69, A80, and I92. A temperature-dependent effect of FapA was observed on FapC by thioflavin T (ThT) and NMR experiments. This study provides a detailed understanding of the interaction between the FapA and FapC, shedding light on the regulation and slowing down of amyloid formation, and has important implications for the development of therapeutic strategies targeting biofilms and associated infections.

Tandem repeats of highly bioluminescent NanoLuc are refolded noncanonically by the Hsp70 machinery.

Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc-based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat-denatured Nluc dyads and triads can be efficiently refolded by the E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co-chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins.

Drugs targeting the particle for arrangement of quaternary structure (PAQosome) and protein complex assembly.

INTRODUCTION: The PAQosome is a 12-subunit complex that acts as a co-factor of the molecular chaperones HSP90 and HSP70. This co-chaperone has been shown to participate in assembly and maturation of several protein complexes, including nuclear RNA polymerases, RNA processing factors, the ribosome, PIKKs, and others. Subunits of the PAQosome, adaptors, and clients have been reported to be involved in various diseases, making them interesting targets for drug discovery. AREA COVERED: In this review, the authors cover the detailed mechanisms of PAQosome and chaperone function. Specifically, the authors summarize the status of the PAQosome and some related chaperones and co-chaperones as candidate targets for drug discovery. Indeed, a number of compounds are currently being tested for the development of treatments against diseases, such as cancers and neurodegenerative conditions. EXPERT OPINION: Searching for new drugs targeting the PAQosome requires a better understanding of PAQosome subunit interactions and the discovery of new interaction partners. Thus, PAQosome subunit crystallization is an important experiment to initiate virtual screening against new target and the development of in silico tools such as AlphaFold-multimer could accelerate the search for new interaction partner and determine more rapidly the interaction pocket needed for virtual drug screening.

Membrane protein chaperone and sodium chloride modulate the kinetics and morphology of amyloid beta aggregation.

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins. Amyloid beta (Aß) peptides are derived from the cleavage of a larger membrane protein molecule and accumulate to form plaques extracellularly. According to the amyloid hypothesis, accumulation of Aß aggregates in the brain is primarily responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, the disassembly of Aß aggregates may provide opportunities for alleviating or treating AD. Here, we show that the novel protein targeting machinery from chloroplast, chloroplast signal recognition particle 43 (cpSRP43), is an ATP-independent membrane protein chaperone that can both prevent and reverse Aß aggregation effectively. Using of thioflavin T dye, we obtained the aggregation kinetics of Aß aggregation and determined that the chaperone prevents Aß aggregation in a concentration-dependent manner. Size exclusion chromatography and sedimentation assays showed that 10-fold excess of cpSRP43 can keep Aß in the soluble monomeric form. Electron microscopy showed that the fibril structure was disrupted in the presence of this chaperone. Importantly, cpSRP43 utilizes the binding energy to actively remodel the preformed Aß aggregates without assistance by a co-chaperone and ATP, emphasizing its unique function among protein chaperones. Moreover, when sodium chloride concentration is higher than 25 mm, the Aß aggregation rate increases drastically to form tightly associated aggregates and generate more oligomers. Our results demonstrate that the presence of cpSRP43 and low NaCl levels inhibit or retard Aß peptide aggregation, potentially opening new avenues to strategically develop an effective treatment for AD.

Methylglyoxal Induced Modifications to Stabilize Therapeutic Proteins: A Review.

Therapeutic proteins are potent, fast-acting drugs that are highly effective in treating various conditions. Medicinal protein usage has increased in the past 10 years, and it will evolve further as we better understand disease molecular pathways. However, it is associated with high processing costs, limited stability, difficulty in being administered as an oral medication, and the inability of large proteins to penetrate tissue and reach their target locations. Many methods have been developed to overcome the problems with the stability and chaperone activity of therapeutic proteins, viz., the addition of external agents (changing the properties of the surrounding solvent by using stabilizing excipients, e.g., amino acids, sugars, polyols) and internal agents (chemical modifications that influence its structural properties, e.g., mutations, glycosylation). However, these methods must completely clear protein instability and chaperone issues. There is still much work to be done on finetuning chaperone proteins to increase their biological efficacy and stability. Methylglyoxal (MGO), a potent dicarbonyl compound, reacts with proteins and forms covalent cross-links. Much research on MGO scavengers has been conducted since they are known to alter protein structure, which may result in alterations in biological activity and stability. MGO is naturally produced within our body, however, its impact on chaperones and protein stability needs to be better understood and seems to vary based on concentration. This review highlights the efforts of several research groups on the effect of MGO on various proteins. It also addresses the impact of MGO on a client protein, α-crystallin, to understand the potential solutions to the protein's chaperone and stability problems.

Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone.

In bacterial flagellum biogenesis, secretion of the hook-filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT, nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homolog of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α-helices and resembles the FliT chaperone, sharing a similar substrate-binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126-144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size-exclusion chromatography and multi-angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled-coil interface between the third helix of FlgN and the C-terminal helix of the substrate. A FlgNΔ126-144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126-144 , like FliT, binds with sub-micromolar affinity to the flagellum ATPase FliI or its N-terminal domain. Hence FlgN and FliT likely couple delivery of low-abundance export substrates to the flagellum ATPase FliI.

Structural characterization of the human DjC20/HscB cochaperone in solution.

J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.