RESUMEN
We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.
Asunto(s)
Antibacterianos , Chaperonina 10 , Escherichia coli , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Chaperonina 10/metabolismo , Chaperonina 10/antagonistas & inhibidores , Chaperonina 10/química , Escherichia coli/efectos de los fármacos , Chaperonina 60/metabolismo , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/química , Benzoxazoles/química , Benzoxazoles/farmacología , Benzoxazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/químicaRESUMEN
In the densely populated intracellular milieu, polypeptides are at constant risk of nonspecific interactions and aggregation, posing a threat to essential cellular functions. Cells rely on a network of protein folding factors to deal with this challenge. The Hsp60 family of molecular chaperones, which depend on ATP for function, stands out in the proteostasis network by a characteristic structure comprising two multimeric rings arranged back to back. This review provides an updated overview of GroEL, the bacterial Hsp60, and its GroES (Hsp10) cofactor. Specifically, we highlight recent breakthroughs in understanding the intricate folding mechanisms of the GroEL-GroES nanomachine and explore the newly discovered interaction between GroEL and the chaperedoxin CnoX. Despite considerable research on the GroEL-GroES system, numerous questions remain to be explored.
Asunto(s)
Chaperonina 10 , Chaperonina 60 , Pliegue de Proteína , Chaperonina 60/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 10/metabolismo , Chaperonina 10/química , Unión Proteica , Bacterias/metabolismo , Bacterias/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genéticaRESUMEN
Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.
Asunto(s)
Adenosina Trifosfato , Chaperonina 10 , Microscopía por Crioelectrón , Modelos Moleculares , Unión Proteica , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Chaperonina 10/metabolismo , Chaperonina 10/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Adenilil Imidodifosfato/metabolismo , Adenilil Imidodifosfato/química , Conformación Proteica , Hydrogenophilaceae/metabolismo , Hydrogenophilaceae/química , Subunidades de Proteína/metabolismo , Subunidades de Proteína/químicaRESUMEN
The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP·BeF3, and GroEL-ADP·AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP·BeF3 to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP·BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, suggesting how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP·AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.
Asunto(s)
Adenosina Trifosfato , Ribulosa-Bifosfato Carboxilasa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Adenosina Trifosfato/metabolismo , Chaperonina 60/metabolismo , Chaperonina 10/química , Pliegue de Proteína , Unión ProteicaRESUMEN
Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, "holding-like" mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differential scanning calorimetry (DSC), light scattering, and circular dichroism. The results showed that the protein in monomeric form is less stable, but more effective against amyloid fibrillization. This greater functionality is attributed to the disordered nature of the domains involved in subunit contacts.
Asunto(s)
Adenosina Trifosfato , Chaperonina 60 , Chaperonina 60/metabolismo , Adenosina Trifosfato/metabolismo , Chaperonina 10/química , Pliegue de ProteínaRESUMEN
Co-chaperonins function together with chaperonins to mediate ATP-dependent protein folding in a variety of cellular compartments. Chaperonins are evolutionarily conserved and form two distinct classes, namely, group I and group II chaperonins. GroEL and its co-chaperonin GroES form part of group I and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo conformational rearrangements that enable protein folding to occur. GroES forms a lid over the chamber and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. Group II chaperonins are functionally similar to group I chaperonins but differ in structure and do not require a co-chaperonin. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances, co-chaperonins display contrasting functions to those of chaperonins. Human HSP60 (HSPD) continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10 on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.
Asunto(s)
Chaperonina 10 , Chaperoninas , Humanos , Chaperonina 10/química , Chaperoninas/química , Chaperoninas/metabolismo , Chaperonina 60/química , Pliegue de Proteína , Chaperoninas del Grupo II/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.
Asunto(s)
Sitio Alostérico , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm â¼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.
Asunto(s)
Adenosina Trifosfato/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Magnesio/metabolismo , Chaperonina 10/química , Chaperonina 60/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligandos , Espectrometría de Masas , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Desplegamiento Proteico , TemperaturaRESUMEN
A protocol for increasing soluble protein expression by fusing the chaperone GroEL apical domain with a gene of interest is described herein. GroEL apical domain, the minichaperone that functions independently of GroES and ATP in protein folding, is cloned downstream of the lambda CII ribosome binding site in the parent pRE vector. The pRE vector has tightly controlled transcription suitable for expressing toxic proteins. The GroEL minichaperone is fused to a glycine-serine rich linker followed by the enterokinase protease recognition sequence. A number of genes that are recalcitrant to protein production in the parent pRE vector 5were cloned into the pRE:GroEL fusion vector and successfully expressed as fusion proteins in Escherichia coli.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Pliegue de ProteínaRESUMEN
The GroEL-GroES chaperonin complex is a bacterial protein folding system, functioning in an ATP-dependent manner. Upon ATP binding and hydrolysis, it undergoes multiple stages linked to substrate protein binding, folding and release. Structural methods helped to reveal several conformational states and provide more information about the chaperonin functional cycle. Here, using cryo-EM we resolved two nucleotide-bound structures of the bullet-shaped GroEL-GroES1 complex at 3.4 Å resolution. The main difference between them is the relative orientation of their apical domains. Both structures contain nucleotides in cis and trans GroEL rings; in contrast to previously reported bullet-shaped complexes where nucleotides were only present in the cis ring. Our results suggest that the bound nucleotides correspond to ADP, and that such a state appears at low ATP:ADP ratios.
Asunto(s)
Adenosina Difosfato/química , Chaperonina 10/química , Chaperonina 60/química , Proteínas de Escherichia coli/química , Adenosina Difosfato/metabolismo , Sitios de Unión , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Microscopía por Crioelectrón , Proteínas de Escherichia coli/metabolismo , Unión ProteicaRESUMEN
Human mitochondrial chaperonin mHsp60 is essential for mitochondrial function by assisting folding of mitochondrial proteins. Unlike the double-ring bacterial GroEL, mHsp60 exists as a heptameric ring that is unstable and dissociates to subunits. The structural dynamics has been implicated for a unique mechanism of mHsp60. We purified active heptameric mHsp60, and determined a cryo-EM structure of mHsp60 heptamer at 3.4 Å. Of the three domains, the equatorial domains contribute most to the inter-subunit interactions, which include a four-stranded ß sheet. Our structural comparison with GroEL shows that mHsp60 contains several unique sequences that directly decrease the sidechain interactions around the ß sheet and indirectly shorten ß strands by disengaging the backbones of the flanking residues from hydrogen bonding in the ß strand conformation. The decreased inter-subunit interactions result in a small inter-subunit interface in mHsp60 compared to GroEL, providing a structural basis for the dynamics of mHsp60 subunit association. Importantly, the unique sequences are conserved among higher eukaryotic mitochondrial chaperonins, suggesting the importance of structural dynamics for eukaryotic chaperonins. Our structural comparison with the single-ring mHsp60-mHsp10 shows that upon mHsp10 binding the shortened inter-subunit ß sheet is restored and the overall inter-subunit interface of mHsp60 increases drastically. Our structural basis for the mHsp10 induced stabilization of mHsp60 subunit interaction is consistent with the literature that mHsp10 stabilizes mHsp60 quaternary structure. Together, our studies provide structural bases for structural dynamics of the mHsp60 heptamer and for the stabilizing effect of mHsp10 on mHsp60 subunit association.
Asunto(s)
Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Proteínas Gestacionales/química , Proteínas Gestacionales/metabolismo , Factores Supresores Inmunológicos/química , Factores Supresores Inmunológicos/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
This review contains a personal account of the role played by the PDB in the development of the field of molecular chaperones and protein homeostasis, from the viewpoint of someone who experienced the concurrent advances in the structural biology, electron microscopy, and chaperone fields. The emphasis is on some key structures, including those of Hsp70, GroEL, Hsp90, and small heat shock proteins, that were determined as the molecular chaperone concept and systems for protein quality control were emerging. These structures were pivotal in demonstrating how seemingly nonspecific chaperones could assist the specific folding pathways of a variety of substrates. Moreover, they have provided mechanistic insights into the ATPase machinery of complexes such as GroEL/GroES that promote unfolding and folding and the disaggregases that extract polypeptides from large aggregates and disassemble amyloid fibers. The PDB has provided a framework for the current success in curating, evaluating, and distributing structural biology data, through both the PDB and the EMDB.
Asunto(s)
Chaperonina 10 , Chaperonina 60 , Bases de Datos de Proteínas , Proteínas HSP70 de Choque Térmico , Proteínas HSP90 de Choque Térmico , Proteolisis , Animales , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , HumanosRESUMEN
Cryo-electron tomography provides the opportunity for unsupervised discovery of endogenous complexes in situ. This process usually requires particle picking, clustering and alignment of subtomograms to produce an average structure of the complex. When applied to heterogeneous samples, template-free clustering and alignment of subtomograms can potentially lead to the discovery of structures for unknown endogenous complexes. However, such methods require scoring functions to measure and accurately rank the quality of aligned subtomogram clusters, which can be compromised by contaminations from misclassified complexes and alignment errors. Here, we provide the first study to assess the effectiveness of more than 15 scoring functions for evaluating the quality of subtomogram clusters, which differ in the amount of structural misalignments and contaminations due to misclassified complexes. We assessed both experimental and simulated subtomograms as ground truth data sets. Our analysis showed that the robustness of scoring functions varies largely. Most scores were sensitive to the signal-to-noise ratio of subtomograms and often required Gaussian filtering as preprocessing for improved performance. Two scoring functions, Spectral SNR-based Fourier Shell Correlation and Pearson Correlation in the Fourier domain with missing wedge correction, showed a robust ranking of subtomogram clusters without any preprocessing and irrespective of SNR levels of subtomograms. Of these two scoring functions, Spectral SNR-based Fourier Shell Correlation was fastest to compute and is a better choice for handling large numbers of subtomograms. Our results provide a guidance for choosing an accurate scoring function for template-free approaches to detect complexes from heterogeneous samples.
Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Imagenología Tridimensional/métodos , Chaperonina 10/química , Chaperonina 60/química , Bases de Datos de Proteínas , Distribución Normal , Ribosomas/química , Relación Señal-RuidoRESUMEN
The Escherichia coli ATP-consuming chaperonin machinery, a complex between GroEL and GroES, has evolved to facilitate folding of substrate proteins (SPs) that cannot do so spontaneously. A series of kinetic experiments show that the SPs are encapsulated in the GroEL/ES nanocage for a short duration. If confinement of the SPs is the mechanism by which GroEL/ES facilitates folding, it follows that the assisted folding rate, relative to the bulk value, should always be enhanced. Here, we show that this is not the case for the folding of rhodanese in the presence of the full machinery of GroEL/ES and ATP. The assisted folding rate of rhodanese decreases. On the basis of our finding and those reported in other studies, we suggest that the ATP-consuming chaperonin machinery has evolved to optimize the product of the folding rate and the yield of the folded SPs on the biological time scale. Neither the rate nor the yield is separately maximized.
Asunto(s)
Chaperonina 10/química , Chaperonina 60/química , Pliegue de Proteína , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Conformación ProteicaRESUMEN
As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coligroESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coligroESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.
Asunto(s)
Chaperonina 10/genética , Chaperonina 60/genética , Escherichia coli/genética , Bacterias Gramnegativas/genética , Bacterias Grampositivas/genética , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Antibacterianos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Enterobacter/efectos de los fármacos , Enterobacter/genética , Enterobacter/metabolismo , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/metabolismo , Cinética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to â¼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of â¼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials.
Asunto(s)
Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Nanopartículas del Metal/química , Adenosina Trifosfato/metabolismo , Chaperonina 60/genética , Chaperoninas/química , Chaperoninas/genética , Chaperoninas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Oro/química , Oro/metabolismo , Hierro/química , Hierro/metabolismo , Microscopía Electrónica de Transmisión de Rastreo , Microscopía Electrónica de Transmisión , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Platino (Metal)/química , Platino (Metal)/metabolismo , Unión Proteica , Pliegue de ProteínaRESUMEN
mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF3-bound) ground-state mimic double-ring mHsp6014-(mHsp107)2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp6014-(mHsp107)2 complex, and a single-ring mHsp607-mHsp107 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.
Asunto(s)
Chaperonina 10/química , Chaperonina 60/química , Mitocondrias/química , Proteínas Mitocondriales/química , Adenosina Difosfato/química , Adenosina Trifosfato/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Citosol/química , Humanos , Enlace de Hidrógeno , Hidrólisis , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Pliegue de ProteínaRESUMEN
Chaperonins are ubiquitous molecular chaperones found in all domains of life. They form ring-shaped complexes that assist in the folding of substrate proteins in an ATP-dependent reaction cycle. Key to the folding cycle is the transient encapsulation of substrate proteins by the chaperonin. Here we present a structural and functional characterization of the chaperonin gp146 (ɸEL) from the phage EL of Pseudomonas aeruginosa. ɸEL, an evolutionarily distant homolog of bacterial GroEL, is active in ATP hydrolysis and prevents the aggregation of denatured protein in a nucleotide-dependent manner. However, ɸEL failed to refold the encapsulation-dependent model substrate rhodanese and did not interact with E. coli GroES, the lid-shaped co-chaperone of GroEL. ɸEL forms tetradecameric double-ring complexes, which dissociate into single rings in the presence of ATP. Crystal structures of ɸEL (at 3.54 and 4.03 Å) in presence of ATPâ¢BeFx revealed two distinct single-ring conformational states, both with open access to the ring cavity. One state showed uniform ATP-bound subunit conformations (symmetric state), whereas the second combined distinct ATP- and ADP-bound subunit conformations (asymmetric state). Cryo-electron microscopy of apo-ɸEL revealed a double-ring structure composed of rings in the asymmetric state (3.45 Å resolution). We propose that the phage chaperonin undergoes nucleotide-dependent conformational switching between double- and single rings and functions in aggregation prevention without substrate protein encapsulation. Thus, ɸEL may represent an evolutionarily more ancient chaperonin prior to acquisition of the encapsulation mechanism.
Asunto(s)
Chaperoninas/química , Pliegue de Proteína , Fagos Pseudomonas/química , Pseudomonas aeruginosa/virología , Proteínas Virales/química , Chaperonina 10/química , Chaperonina 60/química , Microscopía por Crioelectrón , Escherichia coli/química , Proteínas de Escherichia coli/química , Dominios Proteicos , Fagos Pseudomonas/metabolismoRESUMEN
The chaperonin system GroEL-GroES is present in all kingdoms of life and rescues proteins from improper folding and aggregation upon internal and external stress conditions, including high temperatures and pressures. Here, we set out to explore the thermo- and piezostability of GroEL, GroES and the GroEL-GroES complex in the presence of cosolvents, nucleotides and salts employing quantitative FTIR spectroscopy and small-angle X-ray scattering. Owing to its high biological relevance and lack of data, our focus was especially on the effect of pressure on the chaperonin system. The experimental results reveal that the GroEL-GroES complex is remarkably temperature stable with an unfolding temperature beyond 70 °C, which can still be slightly increased by compatible cosolutes like TMAO. Conversely, the pressure stability of GroEL and hence the GroEL-GroES complex is rather limited and much less than that of monomeric proteins. Whereas GroES is pressure stable up to â¼5 kbar, GroEl and the GroEl-GroES complex undergo minor structural changes already beyond 1 kbar, which can be attributed to a dissociation-induced conformational drift. Quite unexpectedly, no significant unfolding of GroEL is observed even up to 10 kbar, however, i.e., the subunits themselves are very pressure stable. As for the physiological relevance, the structural integrity of the chaperonin system is retained in a relatively narrow pressure range, from about 1 to 1000 bar, which is just the pressure range encountered by life on Earth.
Asunto(s)
Chaperonina 10/química , Chaperonina 60/química , Ambiente , Presión , Estabilidad Proteica , TemperaturaRESUMEN
BACKGROUND: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy. METHODS: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever. RESULTS: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated. CONCLUSIONS: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research. GENERAL SIGNIFICANCE: We achieved an imaging rate of ~3â¯s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39â¯nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.