Chaperonin GroEL Research Articles

Immunopeptidomics of Salmonella enterica Serovar Typhimurium-Infected Pig Macrophages Genotyped for Class II Molecules.

Salmonellosis is a zoonotic infection that has a major impact on human health; consuming contaminated pork products is the main source of such infection. Vaccination responses to classic vaccines have been unsatisfactory; that is why peptide subunit-based vaccines represent an excellent alternative. Immunopeptidomics was used in this study as a novel approach for identifying antigens coupled to major histocompatibility complex class II molecules. Three homozygous individuals having three different haplotypes (Lr-0.23, Lr-0.12, and Lr-0.21) were thus selected as donors; peripheral blood macrophages were then obtained and stimulated with Salmonella typhimurium (MOI 1:40). Although similarities were observed regarding peptide length distribution, elution patterns varied between individuals; in total, 1990 unique peptides were identified as follows: 372 for Pig 1 (Lr-0.23), 438 for Pig 2 (Lr.0.12) and 1180 for Pig 3 (Lr.0.21). Thirty-one S. typhimurium unique peptides were identified; most of the identified peptides belonged to outer membrane protein A and chaperonin GroEL. Notably, 87% of the identified bacterial peptides were predicted in silico to be elution ligands. These results encourage further in vivo studies to assess the immunogenicity of the identified peptides, as well as their usefulness as possible protective vaccine candidates.

Characterization of Escherichia coli chaperonin GroEL as a ribonuclease

Chaperonins are evolutionarily conserved proteins that facilitate polypeptide assemblies. The most extensively studied chaperonin is GroEL, which plays a crucial role in Escherichia coli. In addition to its chaperone activity, the RNA cleavage activity of GroEL has also been proposed. However, direct evidence of GroEL as a ribonuclease (RNase) and its physiological significance has not been fully elucidated. Here, we characterized the role of GroEL in E. coli as an RNase distinct from RNase E/G activity using in vivo reporter assays, in vitro cleavage assays with varying reaction times, divalent ions, and 5′ phosphorylation status. GroEL bound to single-stranded RNA at nanomolar concentrations. Functional analysis of GroEL chaperonin-defective mutants and segments identified specific regions, and the chaperone active status of GroEL is not a necessary factor for RNase activity. Additionally, RNase activity of GroEL was attenuated by co-overexpression with GroES. Finally, we characterized potential transcripts regulated by GroEL and the conserved RNase activity of GroEL in Shigella flexneri. Our findings indicate that GroEL is a novel post-transcriptional regulator in bacteria.

To 200,000 m/z and Beyond: Native Electron Capture Charge Reduction Mass Spectrometry Deconvolves Heterogeneous Signals in Large Biopharmaceutical Analytes.

Great progress has been made in the detection of large biomolecular analytes by native mass spectrometry; however, characterizing highly heterogeneous samples remains challenging due to the presence of many overlapping signals from complex ion distributions. Electron-capture charge reduction (ECCR), in which a protein cation captures free electrons without apparent dissociation, can separate overlapping signals by shifting the ions to lower charge states. The concomitant shift to higher m/z also facilitates the exploration of instrument upper m/z limits if large complexes are used. Here we perform native ECCR on the bacterial chaperonin GroEL and megadalton scale adeno-associated virus (AAV) capsid assemblies on a Q Exactive UHMR mass spectrometer. Charge reduction of AAV8 capsids by up to 90% pushes signals well above 100,000 m/z and enables charge state resolution and mean mass determination of these highly heterogeneous samples, even for capsids loaded with genetic cargo. With minor instrument modifications, the UHMR instrument can detect charge-reduced ion signals beyond 200,000 m/z. This work demonstrates the utility of ECCR for deconvolving heterogeneous signals in native mass spectrometry and presents the highest m/z signals ever recorded on an Orbitrap instrument, opening up the use of Orbitrap native mass spectrometry for heavier analytes than ever before.

Visualizing chaperonin function in situ by cryo-electron tomography

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1–3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4–6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55–70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL–GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL–GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Genome Sequence Resource of Bacillus mojavensis KRS009, a Rhizospheric Microorganism with Biocontrol Potential

Bacillus mojavensis KRS009 was identified as an antagonistic strain with a strong inhibitory effect on various phytopathogenic fungi. To provide further insight into its biocontrol mechanisms and ability to improve plant salt tolerance, the high quality complete genome of KRS009 was sequenced by PacBio platform and assembled using PacBio-HiFiReads. The KRS009 genome consists of one circular chromosome of 4,089,687 bp with 43.5% GC content and 4,062 open reading frames, of which 3,920 protein-coding genes, 86 transfer RNA, 10 ribosomal RNA, and 26 small RNA were identified in this genome. Among them, genes related to compatible solutes, including treC, galU, proX, and proW, are involved in the synthesis of trehalose, proline, and betaine metabolism and play an important role in relieving osmotic stress. In addition, the KRS009 genome contains genes associated with high salinity tolerance, including those responsible for Na+/H+ antiporters, K+ transporters, TrkH thiamin phosphate synthase, K+-sensing histidine kinase, aryl-phospho-beta-D-glucosidase, flavoprotein CzcO associated with the cation diffusion facilitator CzcD, and chaperonin GroEL. Together, the high-quality genome resource of strain KRS009 would provide a molecular basis for further research on its biocontrol and plant salt tolerance mechanisms. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Distribution and Potential Ecophysiological Roles of Multiple GroEL Chaperonins in Pink-Pigmented Facultative Methylotrophs

Distribution and Potential Ecophysiological Roles of Multiple GroEL Chaperonins in Pink-Pigmented Facultative Methylotrophs

Beyond copper: examining the significance of His-mutations in mycobacterial GroEL1 HRCT for Ni(II) complex stability and formation.

Recently, we have studied the coordination chemistry of the Cu(II)-histidine-rich C-terminal tail (HRCT) complex of the mycobacterial GroEL1 protein. The structure of this domain differs significantly compared to the well-known methionine-glycine-rich GroEL chaperonin - it was predicted that mycobacterial GroEL1 could play a significant role in the metal homeostasis of Mycobacteria, especially copper. However, we found that this particular domain's pattern also repeats in a number of Ni(II)-binding proteins. Here, we present the studies concerning the properties of GroEL1 HRCT as a ligand for Ni(II) ions. For this purpose, we chose eight model peptides: L1 - Ac-DHDHHHGHAH, L2 - Ac-DKPAKAEDHDHHHGHAH, and 6 mutants of the latter in the pH range of 2-11. We examined the stoichiometry, stability, and spectroscopic features of copper complexes. We noticed that similar to the Cu(II)-complex, the presence of a Lys5 residue significantly increases the stability of the system. The impact of His mutations was also examined and carefully studied using NMR spectroscopy. His9 and His13 are the crucial residues for Ni(II) binding, whereas His12 has minimal relevance in complex formation.

Exploring binding preferences: Cu(II), Ni(II), and Zn(II) complexes of mycobacterial GroEL1 His-rich and Glu/His-rich domains.

Mycobacterial histidine-rich GroEL1 protein significantly differs from the well-known methionine-glycine-rich GroEL chaperonin and most preferably participates in Cu(II) homeostasis. Some GroEL1 proteins, however, do not possess six but only three histidine residues and more acidic residues that can function as binding sites for metal ions. To evaluate the importance of this difference, we examined and compared the properties of GroEL1 His-rich or Glu/His-rich C-terminal domains as ligands for Cu(II), Ni(II), and Zn(II) ions. We studied the stoichiometry, stability, and binding sites of Cu(II)/Ni(II)/Zn(II) complexes of two model peptides: XEN = Ac-DKPEEEEDGHGHAH (M. xenopi) and ABS = Ac-DKPAEEADHGHGHHGHAH (M. abscessus) in the pH range 2-11. In the case of Cu(II), Ni(II), and Zn(II) complexes of XEN and ABS, ABS always formed more stable complexes. For XEN, there seemed to be no preference for Ni(II) or Zn(II) ions. In contrast, for ABS, Zn(II) formed a complex that was slightly more stable than the one formed by Ni(II). This may be due to the 6 His residues, which preferentially interact with Zn(II) rather than Ni(II). The study identified that an equilibrium of complexes-known as polymorphism-may occur in ABS complexes. Therefore, distinct sets of histidine residues may be involved in metal binding.

From Microstates to Macrostates in the Conformational Dynamics of GroEL: A Single-Molecule Förster Resonance Energy Transfer Study.

The chaperonin GroEL is a multisubunit molecular machine that assists in protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. Here, we report single-molecule Förster resonance energy transfer measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the cochaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-ligated conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis. These coinciding results from bulk measurements for an entire ring and single-molecule measurements for a single subunit provide new evidence for the concerted allosteric transition of all seven subunits.

ATP-Responsive Nanoparticles Covered with Biomolecular Machine "Chaperonin GroEL".

Herein, we report an ATP-responsive nanoparticle (GroELNP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroELNP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroELNPs was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable GroELNPs to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroELNPs per GroEL was 4.8 and 4.0 times greater than those of precursor cysGroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that GroELNPs could be iteratively extended to double-layered (GroEL)2NPs.

ATP‐Responsive Nanoparticles Covered with Biomolecular Machine “Chaperonin GroEL”

AbstractHerein, we report an ATP‐responsive nanoparticle (GroELNP) whose surface is fully covered with the biomolecular machine “chaperonin protein GroEL”. GroELNP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroELNP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine‐like function and enable GroELNP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroELNP per GroEL was 4.8 and 4.0 times greater than those of precursor cysGroEL and its DNA‐functionalized analogue, respectively. Finally, we confirmed that GroELNP could be iteratively extended to double‐layered NP.

Potential contribution of Helicobacter pylori proteins in the pathogenesis of type 1 gastric neuroendocrine tumor and urticaria. In silico approach.

Helicobacter pylori has been linked to several diseases such as chronic urticaria, gastritis, and type 1 gastric neuroendocrine tumors (type 1 gNET). Although these diseases seem to have different mechanisms, their relationship with H. pylori suggests a common inflammatory pathway. To identify potential cross-reactive antigens between H. pylori and humans involved in chronic urticaria and type 1 gNET. Alignment was carried out among human proteins associated with urticaria (9 proteins), type 1 gNET (32 proteins), and H. pylori proteome. We performed pairwise alignment among the human and H. pylori antigens with PSI-BLAST. Modeling based on homology was done with the Swiss model server and epitope prediction with the Ellipro server. Epitopes were located on a 3D model using PYMOL software. The highest conserved sequence was found between the human HSP 60 antigen and the H. pylori chaperonin GroEL with an identity of 54% and a cover of 92%, followed by the alpha and gamma enolases and two H. pylori phosphopyruvate hydratase, both with an identity and cover of 48% and 96%, respectively. The H/K ATPase (Chain A) showed high identity with two H. pylori proteins (35.21% with both P-type ATPase), but with low cover (only 6%). We observed eight linear and three discontinuous epitopes for human HSP 60 and three lineal and one discontinuous epitope for both alpha-enolase and gamma enolase, high conserved with H. pylori sequences. Some type 1 gNET antigens shared potential cross-reactive epitopes with H. pylori proteins, suggesting that molecular mimicry could be a mechanism that explains the relationship between the infection and this disease. Studies evaluating the functional impact of this relationship are needed.

Histidine-Rich C-Terminal Tail of Mycobacterial GroEL1 and Its Copper Complex─The Impact of Point Mutations

The mycobacterial histidine-rich GroEL1 protein differs significantly compared to the well-known methionine/glycine-rich GroEL chaperonin. It was predicted that mycobacterial GroEL1 can play a significant role in the metal homeostasis of Mycobacteria but not, as its analogue, in protein folding. In this paper, we present the properties of the GroEL1 His-rich C-terminus as a ligand for Cu(II) ions. We studied the stoichiometry, stability, and spectroscopic features of copper complexes of the eight model peptides: L1─Ac-DHDHHHGHAH, L2─Ac-DKPAKAEDHDHHHGHAH, and six mutants of L2 in the pH range of 2-11. We revealed the impact of adjacent residues to the His-rich fragment on the complex stability: the presence of Lys and Asp residues significantly increases the stability of the system. The impact of His mutations was also examined: surprisingly, the exchange of each single His to the Gln residue did not disrupt the ability of the ligand to provide three binding sites for Cu(II) ions. Despite the most possible preference of the Cu(II) ion for the His9-His13 residues (Ac-DKPAKAEDHDHHH-) of the model peptide, especially the His11 residue, the study shows that there is not only one possible binding mode for Cu(II). The significance of this phenomenon is very important for the GroEL1 function─if the single mutation occurs naturally, the protein would be still able to interact with the metal ion.

Predicting allosteric sites using fast conformational sampling as guided by coarse-grained normal modes.

To computationally identify cryptic binding sites for allosteric modulators, we have developed a fast and simple conformational sampling scheme guided by coarse-grained normal modes solved from the elastic network models followed by atomistic backbone and sidechain reconstruction. Despite the complexity of conformational changes associated with ligand binding, we previously showed that simply sampling along each of the lowest 30 modes can adequately restructure cryptic sites so they are detectable by pocket finding programs like Concavity. Here, we applied this method to study four classical examples of allosteric regulation (GluR2 receptor, GroEL chaperonin, GPCR, and myosin). Our method along with alternative methods has been utilized to locate known allosteric sites and predict new promising allosteric sites. Compared with other sampling methods based on extensive molecular dynamics simulation, our method is both faster (1-2h for an average-size protein of ∼400 residues) and more flexible (it can be easily integrated with any structure-based pocket finding methods), so it is suitable for high-throughput screening of large datasets of protein structures at the genome scale.

Prediction of chaperonin GroE substrates using small structural patterns of proteins.

Molecular chaperones are indispensable proteins that assist the folding of aggregation-prone proteins into their functional native states, thereby maintaining organized cellular systems. Two of the best characterized chaperones are the Escherichia coli chaperonins GroEL and GroES (GroE), for which in vivo obligate substrates have been identified by proteome-wide experiments. These substrates comprise various proteins, but exhibit remarkable structural features. They include a number of α/β proteins, particularly those adopting the TIM β/α barrel fold. This observation led us to speculate that GroE obligate substrates share a structural motif. Based on this hypothesis, we exhaustively compared substrate structures with the MICAN alignment tool, which detects common structural patterns while ignoring the connectivity or orientation of secondary structural elements. We selected four (or five) substructures with hydrophobic indices that were mostly included in substrates and excluded in others, and developed a GroE obligate substrate discriminator. The substructures are structurally similar and superimposable on the 2-layer 2α4β sandwich, the most popular protein substructure, implying that targeting this structural pattern is a useful strategy for GroE to assist numerous proteins. Seventeen false positives predicted by our methods were experimentally examined using GroE-depleted cells, and 9 proteins were confirmed to be novel GroE obligate substrates. Together, these results demonstrate the utility of our common-substructure hypothesis and prediction method.

A molecular device for the redox quality control of GroEL/ES substrates.

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties.

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

DNA starvation/stationary phase protection protein of Helicobacter pylori as a potential immunodominant antigen for infection detection.

Application of chicken egg yolk immunoglobulin Y (IgY) for Helicobacter pylori (H.pylori, HP) has gained much interest in recent years. Comparing with for treatment, IgY may be more advantageous when used for H.pylori detection. Nine strains of H.pylori with different genetic backgrounds were inactivated and used to immunize hens, respectively, for the preparation of polyclonal anti-H.pylori immunoglobulin Y (anti-HP IgY). The proteins of H.pylori with reactivity to anti-HP IgY were detected by Western Blot. The five protein bands that can be well recognized by anti-HP IgY of each group, and were prevalent in all nine strains were excised from SDS-PAGE gel, digested and identified by Nano-HPLC-MS/MS analysis. The potential of these identified proteins as antigen detection targets was then assessed by sequence analysis. Anti-HP IgY derived from each group of specific strain immunized hens can recognize self-strain and non-self-strain antigens well. Five immunodominant antigens were identified as chaperonin GroEL, flagellin A, urease subunit alpha, peroxiredoxin and DNA starvation/stationary phase protection protein. Sequences analysis showed that both peroxiredoxin and DNA starvation/stationary phase protection protein were present in all 1000 strains of H.pylori queried, and the amino acid sequences were highly conserved. The highest sequence consistency between the DNA starvation/stationary phase protection protein of H.pylori and non-Helicobacter organisms was 52.59%, and the consistent sites were scattered and there was no continuous long fragment consensus sequence. DNA starvation/stationary phase protection protein was identified as an immunodominant antigen of H.pylori and sequence analysis indicated that it could serve as a potential antigen target for the diagnosis of H.pylori infection.

In vivo client proteins of the chaperonin GroEL-GroES provide insight into the role of chaperones in protein evolution.

Protein folding is often hampered by intermolecular protein aggregation, which can be prevented by a variety of chaperones in the cell. Bacterial chaperonin GroEL is a ring-shaped chaperone that forms complexes with its cochaperonin GroES, creating central cavities to accommodate client proteins (also referred as substrate proteins) for folding. GroEL and GroES (GroE) are the only indispensable chaperones for bacterial viability, except for some species of Mollicutes such as Ureaplasma. To understand the role of chaperonins in the cell, one important goal of GroEL research is to identify a group of obligate GroEL/GroES clients. Recent advances revealed hundreds of in vivo GroE interactors and obligate chaperonin-dependent clients. This review summarizes the progress on the in vivo GroE client repertoire and its features, mainly for Escherichia coli GroE. Finally, we discuss the implications of the GroE clients for the chaperone-mediated buffering of protein folding and their influences on protein evolution.

Chaperonin GroEL hydrolyses ortho-nitrophenyl β-galactoside.

We serendipitously found that chaperonin GroEL can hydrolyze ortho-nitrophenyl β-galactoside (ONPG), a well-known substrate of the enzyme β-galactosidase. The ONPG hydrolysis by GroEL follows typical enzyme kinetics. Our experiments and molecular docking studies suggest ONPG binding at the ATP binding site of GroEL.