Figure 4 - uploaded by Arthur L Horwich
Content may be subject to copyright.
Inter-subunit contacts in the cis ring of the GroEL-GroES complex. a, b, Ribbons drawings for two adjacent subunits in trans (a) and cis (b) GroEL rings viewed from the inside of the ring. In each panel, the left GroEL subunit is yellow and the right subunit is cyan. Two GroES subunits (orange and green) are shown in b, along with two bound ADP molecules (blue). The substructures forming the new interface between equatorial subunits are red, and the substructures forming the GroEL-GroES interface are magenta. c, The site of contact at the new interface between equatorial subunits in the cis ring. The orientation is roughly the same as that of b. Side chains of residues at the contact surface are shown as ball-and-stick models: carbon, white; nitrogen, green; oxygen, red; sulphur, yellow. Van der Waals surfaces are represented by dot drawing. Shortened radii of 1 A ˚ are used for clarity. Backbone traces are represented by a coiled-line ribbon drawing. Both the ribbon and surface are yellow for the left subunit and cyan for the right subunit. Hydrogen bonds are shown as white broken lines.
Source publication
Chaperonins assist protein folding with the consumption of ATP. They exist as multi-subunit protein assemblies comprising rings of subunits stacked back to back. In Escherichia coli, asymmetric intermediates of GroEL are formed with the co-chaperonin GroES and nucleotides bound only to one of the seven-subunit rings (the cis ring) and not to the op...
Contexts in source publication
Context 1
... is cyan, except the 'mobile loop' which is purple. b, Ribbons drawing of one subunit in the unliganded GroEL structure. the neighbouring subunit (helix C). Residues from helix M of the reoriented intermediate domain of the cis ring form contacts with the loop of the stem loop within the same subunit and with helix C of the neighbouring subunit (Fig. 4). In addition, the loop of the stem loop forms a more extensive interaction with a neighbouring helix C. This establishes a structural connection within the cis ring that coordinates the binding of nucleotide and ...
Context 2
... a long loop between strands 6 and 7 (Tyr 199, Ser 201, Tyr 203 and Phe 204) (Fig. 5a). In the GroEL-GroES complex, helices H and I have moved to the top of the subunit to form part of the GroEL-GroES interface, and the loop between strand 6 and strand 7 is elevated and rotated into the cis ring's newly formed interface between apical domains ( Fig. 5b; also Fig. 4a, b). Thus the binding of GroES and nucleotide deprives the substrate polypeptide of its binding elements because the residues in the cis ring implicated in binding non-native polypeptide are now involved in either binding GroES directly (helices H and I) or supporting GroES binding indirectly by stabilizing the interface between elevated ...
Similar publications
We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding
of tetradecameric GroEL from urea-denatured protein monomers. The results show that productive refolding and assembly strongly
depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotid...
Citations
... Like GroEL, the human mHsp60 protein can form stacked double-heptameric rings arranged back-to-back, which are enclosed by a dome-shaped lid consisting of heptameric mHsp10 (homologous to GroES in E. coli). These structures act as ATP-driven nanocages where substrate proteins can fold within a confined space [12,13]. On the contrary, Group II chaperonins are found in archaea and eukaryotes. ...
External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium and ensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP‐dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, β, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αβγ, Hsp60αβ, and Hsp60β, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP‐dependent manner through charge‐driven interactions. Furthermore, the Hsp60β homo‐oligomeric complex can protect the archaeal and eukaryotic membrane from stress‐induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC‐type cooperativity is nested within KNF‐type cooperativity. Remarkably, during ATP hydrolysis, Hsp60β, and Hsp60αβ complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality.
... To perform this plethora of functions, high constitutive expression levels of the chaperonin genes are required, ranking chaperonins among the most abundant cellular proteins. Bacterial chaperonins consist of Cpn60, a homotetradecameric double-ring cylinder composed of 57 kDa subunits and its co-chaperonin Cpn10, a homoheptameric dome-shaped ring composed of approximately 10 kDa subunits (Braig et al. 1994;Hunt et al. 1996;Xu et al. 1997;Saibil et al. 2013). Paradigms for Cpn60 and Cpn10 are the Escherichia coli (Ec) proteins GroEL and GroES, respectively. ...
Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be a beneficial adaptation to the cold environment. Here we show that Cpn60 from the psychrophile Pseudoalteromonas haloplanktis (Ph) maintains its double-ring structure also in the cold. A strongly reduced ATPase activity keeps the chaperonin in an energy-saving dormant state, until binding of client protein activates it. Ph Cpn60 in complex with co-chaperonin Ph Cpn10 efficiently assists in protein folding up to 55 °C. Moreover, we show that recombinant expression of Ph Cpn60 can provide its host Escherichia coli with improved viability under low temperature growth conditions. These properties of the Ph chaperonin may make it a valuable tool in the folding and stabilization of psychrophilic proteins.
... In this research, we employed predictions from the PONDR ® VLXT program to create circular permutations of the apical domain of the GroEL (AD-GroEL) chaperone [25], each exhibiting distinct stability. The GroEL apical domain plays an important role in binding non-native target proteins [26][27][28][29][30][31], a critical aspect of the GroEL chaperone's functionality. When examined in isolation, the 15 kDa GroEL apical domain retains some of its functional characteristics, enabling it to bind nonnative proteins, and is often denoted as a "minichaperone" [28,[32][33][34][35]. ...
Enhancing protein stability holds paramount significance in biotechnology, therapeutics, and the food industry. Circular permutations offer a distinctive avenue for manipulating protein stability while keeping intra-protein interactions intact. Amidst the creation of circular permutants, determining the optimal placement of the new N- and C-termini stands as a pivotal, albeit largely unexplored, endeavor. In this study, we employed PONDR-FIT’s predictions of disorder propensity to guide the design of circular permutants for the GroEL apical domain (residues 191–345). Our underlying hypothesis posited that a higher predicted disorder value would correspond to reduced stability in the circular permutants, owing to the increased likelihood of fluctuations in the novel N- and C-termini. To substantiate this hypothesis, we engineered six circular permutants, positioning glycines within the loops as locations for the new N- and C-termini. We demonstrated the validity of our hypothesis along the set of the designed circular permutants, as supported by measurements of melting temperatures by circular dichroism and differential scanning microcalorimetry. Consequently, we propose a novel computational methodology that rationalizes the design of circular permutants with projected stability.
... Based on X-ray crystallography, electron microscopy (EM), and cryo-EM, a plethora of previously reported studies showed that the conformations of GroEL 14 and GroEL 14 -GroES 7 complexes were dramatically different, following a major conformational change of GroEL 14 , triggered by the nucleotide binding, upon complexation with the co-chaperone GroES 7 . [6,[56][57][58]61,64] We, therefore, attributed the shift of the migration time between GroEL 14 -(ATP S) 14 and GroEL 14 -(ATP S) 7 -GroES 7 complexes to the substantial conformational rearrangement of GroEL 14 upon complexation with the co-chaperone GroES 7 and seven ATP S molecules, under the described CE-MS conditions. The mass difference of 69 kDa between GroEL 14 -(ATP S) 14 and GroEL 14 -(ATP S) 7 -GroES 7 complexes could not explain the significant migration time shift between both complexes since, as described above with the SPbound GroEL 14 complexes, a mass increment of up to 66 kDa did not affect the migration time of the near 1 MDa GroEL 14 protein assembly in the absence of a drastic conformational alteration of the GroEL 14 folding chamber. ...
... The dimensions of mutant GroEL 14 , mutant GroEL 14 -(ATP S) 14 , and mutant GroEL 14 -(ATP S) 7 -GroES 7 complexes were assessed based on the measurement of the computationally designed 3D structures and previously reported studies. [56][57][58]64] The computational modeling of the protein 3D structures included the first methionine residue at the N-termini of the monomeric subunits. Therefore, a shift of +1 in the amino acid numbering is systematically observed compared to the numbering of b and y terminal and by internal fragment ions. ...
Protein complexes are essential forproteins' folding and biological function. Currently, native analysis of largemultimeric protein complexes remains challenging. Structural biology techniquesare time‐consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis‐mass spectrometry (CE–MS) method is reportedto characterize, under near‐physiological conditions, the conformationalrearrangements of ∽1 MDa GroEL upon complexation with binding partners involvedin a protein folding cycle. The developed CE‐MS method is fast (30min per run), highly sensitive (low‐amol level), and requires ∽10 000‐foldfewer samples compared to biochemical/biophysical techniques. The methodsuccessfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73 kDa), and NanA4 (∽130 kDa) oligomers. The non‐covalent binding of naturalsubstrate proteins with GroEL14 can be detected and quantified.The technique allows monitoring of GroEL14 conformational changesupon complexation with (ATPγS)4–14 and GroES7 (∽876 kDa). NativeCE‐pseudo‐MS3 analyses of wild‐type (WT) GroEL and two GroEL mutants resultin up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE–MS performance for multimeric complexes' characterization versus directinfusion ESI–MS. This study shows the CE–MS potential to provide information onbinding stoichiometry and kinetics for various protein complexes.
... Although not surprising, long-lived (approximately 1.5 s) symmetric GroEL-GroES 2 complexes, referred to as football (F) complexes, were frequently observed (97) (Figure 5a). This observation contradicts the prevailing model that asymmetric GroEL-GroES complexes, referred to as bullet (B) complexes, would be exclusively or at least predominantly formed (51,140) due to strong interring negative cooperativity with regard to ATP binding (146). ...
Structural biology is currently undergoing a transformation into dynamic structural biology, which reveals the dynamic structure of proteins during their functional activity to better elucidate how they function. Among the various approaches in dynamic structural biology, high-speed atomic force microscopy (HS-AFM) is unique in the ability to film individual molecules in dynamic action, although only topographical information is acquirable. This review provides a guide to the use of HS-AFM for biomolecular imaging and showcases several examples, as well as providing information on up-to-date progress in HS-AFM technology. Finally, we discuss the future prospects of HS-AFM in the context of dynamic structural biology in the upcoming era.
Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The equatorial and apical domains are linked by intermediate domains that serve as hinges allowing for the rearrangement of large structures during the GroE functional cycle. The GroEL ring forms a cavity that serves as a folding compartment for the polypeptide substrate (Xu et al. 1997). GroES is a cochaperone of GroEL, which has a dome-shaped ring structure and consists of seven subunits. ...
Escherichia coli is widely used as a host for expressing recombinant proteins due to its well-studied genetics, fast growth, relatively low production costs, and high rate of protein expression. However, despite the high rate of protein expression, the availability of chaperone proteins was often insufficient, resulting in the formation of inclusion bodies due to errors in protein folding. These inclusion bodies can cause the protein to become inactive, and proper protein folding is crucial for maintaining the structure and function of proteins in living organisms. To overcome this limitation, chaperones have been developed as a strategy to help prevent protein folding errors and increase the recovery of soluble protein. In this review, we summarize several experiments related to co-expressing chaperones to enhance the expression of recombinant proteins in E. coli. Abstrak: Escherichia coli merupakan bakteri yang paling banyak digunakan sebagai inang pada ekspresi protein rekombinan. Penggunaan Escherichia coli sebagai inang memiliki kelebihan diantaranya, genetikanya telah dipelajari dengan baik, pertumbuhannya cepat, biaya produksi relatif murah, dan laju ekspresi protein yang tinggi. Namun, tingginya laju ekspresi protein pada Escherichia coli tidak diimbangi dengan ketersediaan protein chaperon sehingga terbentuklah badan inklusi yang disebabkan oleh kesalahan pelipatan protein. Terbentuknya badan inklusi dapat menyebabkan protein menjadi tidak aktif. Salah satu strategi yang dikembangkan untuk mengatasi keterbatasan tersebut adalah penggunaan chaperon yang berfungsi untuk membantu mencegah terjadinya kesalahan pelipatan protein serta meningkatkan perolehan protein terlarut. Dalam artikel review ini, akan dipaparkan gambaran umum mengenai pengaruh ko-ekspresi chaperon (GroEL-ES dan Trigger Factor) dalam ekspresi protein rekombinan di Escherichia coli. Artikel ini diharapkan dapat menjadi referensi untuk penggunaan ko-ekspresi chaperon pada ekspresi protein rekombinan secara di Escherichia coli.
... Sequence alignments show differences in the highly conserved equatorial domain residues, particularly at the N-and C-termini ( Figure S8). The inter-subunit interactions that stabilise the GroEL tetradecamer occur mostly between equatorial domain residues, including key residues at the N-terminus (Braig et al., 1994;Xu et al., 1997). Mutating the GroEL A2 (Horovitz et al., 1993) and E76 residues to serines, the corresponding residues in MtbCpn60.1, ...
Mycobacterium tuberculosis encodes two chaperonin proteins, MtbCpn60.1 and MtbCpn60.2, that share substantial sequence similarity with the Escherichia coli chaperonin, GroEL. However, unlike GroEL, MtbCpn60.1 and MtbCpn60.2 purify as lower‐order oligomers. Previous studies have shown that MtbCpn60.2 can functionally replace GroEL in E. coli, while the function of MtbCpn60.1 remained an enigma. Here, we demonstrate the molecular chaperone function of MtbCpn60.1 and MtbCpn60.2, by probing their ability to assist the folding of obligate chaperonin clients, DapA, FtsE and MetK, in an E. coli strain depleted of endogenous GroEL. We show that both MtbCpn60.1 and MtbCpn60.2 support cell survival and cell division by assisting the folding of DapA and FtsE, but only MtbCpn60.2 completely rescues GroEL‐depleted E. coli cells. We also show that, unlike MtbCpn60.2, MtbCpn60.1 has limited ability to support cell growth and proliferation and assist the folding of MetK. Our findings suggest that the client pools of GroEL and MtbCpn60.2 overlap substantially, while MtbCpn60.1 folds only a small subset of GroEL clients. We conclude that the differences between MtbCpn60.1 and MtbCpn60.2 may be a consequence of their intrinsic sequence features, which affect their thermostability, efficiency, clientomes and modes of action.
... GroES, on the other hand, bind to one or both ends of the GroEL complex after forming a heptamer ring driven by ATP. Both Cpns are required for E. coli (Xu et al., 1997;Grallert and Buchner, 2001). But in M. tuberculosis, the situation is different. ...
Nitrogen metabolism is an important physiological process that affects the
survival and virulence of Mycobacterium tuberculosis. M. tuberculosis’s utilization
of nitrogen in the environment and its adaptation to the harsh environment of
acid and low oxygen in macrophages are closely related to nitrogen metabolism.
In addition, the dormancy state and drug resistance of M. tuberculosis are closely
related to nitrogen metabolism. Although nitrogen metabolism is so important,
limited research was performed on nitrogen metabolism as compared with
carbon metabolism. M. tuberculosis can use a variety of inorganic or organic
nitrogen sources, including ammonium salts, nitrate, glutamine, asparagine, etc.
In these metabolic pathways, some enzymes encoded by key genes, such as
GlnA1, AnsP2, etc, play important regulatory roles in the pathogenesis of TB.
Although various small molecule inhibitors and drugs have been developed
for different nitrogen metabolism processes, however, long-term validation is
needed before their practical application. Most importantly, with the emergence
of multidrug-resistant strains, eradication, and control of M. tuberculosis will still
be very challenging.
... First identified as a heat-shock protein (5,6), GroEL is essential for the successful folding of certain proteins in the cell under either normal or stress conditions (7)(8)(9)(10). It consists of 14 identical subunits arranged in two stacked heptameric rings, each forming a folding cavity that can accommodate a non-native protein substrate (11)(12)(13)(14). The GroEL subunit can be segmented into three functional domains (Figure 1 A). ...
... The equatorial domain contains the nucleotide binding pocket (15,16) and forms the inter-ring interface (13,17). The apical domain is involved in the interaction with non-native protein substrates (18)(19)(20) and the cochaperone GroES, a heptameric complex that occludes the folding cage with the substrate protein inside (12). Finally, the intermediate domain connects the equatorial and apical domains and plays an essential role in the activity of the protein (16). ...
... The intra-ring concerted transitions are described by the Monod-Wyman-Changeux (MWC) formalism (32), which posits an equilibrium between two allosteric states, T (tense) and R (relaxed), with low and high affinity towards ATP binding, respectively. X-ray crystallography (11,12,14,33,34) and cryo-EM (13,(35)(36)(37) structures reveal that GroEL samples a rich conformational space with large domain motions during its reaction cycle. Upon ATP binding, the GroEL subunits transition from a closed to an open conformation in an upward motion of its apical domains. ...
The chaperonin GroEL is a multi-subunit molecular machine that assists in protein folding in the E. coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. However, a measurement of subunit dynamics and their relation to the allosteric cycle of GroEL has been missing. Here, we report single-molecule FRET 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 co-chaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-liganded conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis, confirming the essential role of the observed dynamics in the function of GroEL.
Significance Statement
GroEL is a large protein-folding machine whose activity is accompanied by considerable conformational motions. Here, we use single-molecule FRET spectroscopy in combination with photon-by-photon statistical analysis to characterize the motions of a single GroEL subunit in real time and in the presence of ADP, ATP, and the co-chaperone GroES. Our results reveal transitions between four conformations on a timescale much faster than the functional cycle. We show that the motions of an individual subunit are directly coupled to the concerted allosteric mechanism of GroEL. This work, therefore, further demonstrates the impact of fast conformational dynamics on the biochemical function of molecular machines.
... One of the best-studied chaperones is the chaperonin GroEL/GroES (GroE) from E. coli [9][10][11][12], composed of two stacked heptameric rings (GroEL) and a hatchlike cofactor (GroES) [13]. GroEL has regions that bind non-native states of proteins [14][15][16]. ...
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.
