Introduction
It is well established that in vitro certain small globular proteins are able to fold spontaneously into their native state, showing that all the information that defines the three-dimensional structure of a protein is present in its primary sequence (1). However, larger proteins, that typically comprise multiple domains, either fail to fold spontaneously in vitro or this takes too long to be biologically significant. It is therefore not surprising that in the crowded environment of the cell protein folding is assisted by different proteinaceous factors that are commonly known as molecular chaperones (2,3). In general, protein folding resembles a downhill journey through a funnel-shaped energy landscape towards the native state, thereby sampling different energetically stable conformational intermediates (figure 1 adapted from 2). Protein folding is driven by the progressive burial of hydrophobic residues within the core of the protein, which gradually increases the number of native interactions and restricts the conformational space that needs to be sampled to find the native state. These conformational intermediates may accumulate, which, in turn, slows protein folding, and thereby increasing the possibility of kinetically trapping these misfolded species.
These are likely to form thermodynamically stable aggregates (amyloids) that are often cytotoxic and are in humans tightly linked to several progressive neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s and ALS (4). Molecular chaperones inhibit protein aggregation, resolve kinetically trapped intermediates and support protein folding through lowering the energy barriers of folding intermediates en route to the native state. A molecular chaperone represents any protein that associates with, stabilizes or assists another protein to attain its biologically active conformation without being present in the final structure of its client protein (2,3). Owing to their crucial role, molecular chaperones are ubiquitous in all domains of life and are essential for cell viability. Each cell contains a set of different unrelated molecular chaperones that operate in complementary networks to ensure a functional proteome. Chaperones are also known as stress proteins or heat shock proteins (Hsps) because they are upregulated during stress to protect the proteome, thereby maintaining cellular function. They are classified according to their molecular weight, yielding the following families: Hsp40, Hsp60, Hsp70, Hsp90, Hsp100 and the small Hsps. Of these, Hsp70s, Hsp60s (chaperonins) and Hsp90s are multicomponent nanomachines that aid protein (re)folding in an ATP-dependent fashion (2,3). These chaperones typically bind to exposed hydrophobic residues in non-native proteins which temporarily stops aggregation. Subsequently, ATP binding induces release of the chaperone, allowing folding to proceed. Although Hsp70s and Hsp60s function according to the general mechanism outlined above, they differ structurally. Hsp70s release client proteins into solution for folding, while chaperonins comprise a barrel-shaped cage in which a single client protein is allowed to fold (2,3). Recently, detailed structures of the human chaperonin complex were reported (5,6,7), providing important molecular insight into the process of Hsp60-mediated protein folding. Here, I will discuss these structures as well as their current functional understanding.
Protein quality control within the mitochondrial matrix
Human Hsp60 and its cochaperonin Hsp10 are essential for cell viability and both are localized to the mitochondrial matrix. Within this compartment, the Hsp60-Hsp10 complex assists the folding of mitochondrial matrix proteins (8). Importantly, each mitochondrial compartment – the outer membrane, inner membrane space – inner membrane and matrix – houses a set of dedicated chaperones and proteases that are jointly responsible for the quality control of the respective mitochondrial subproteome through ensuring the (re)folding of newly synthesized proteins and removal of misfolded species (8,9). Accumulating evidence shows that mitochondrial activity is strongly linked to the integrity of the mitochondrial proteome. Thus, the mitochondrial quality control system directly controls proper mitochondrial activity, which ranges from ATP production to cofactor biosynthesis as well as the metabolism of nucleotides, lipids and amino acids. Hence, a proper mitochondrial activity is essential for cellular viability and health. In fact, mitochondrial dysfunction is in humans associated with the development of neurodegenerative diseases, cardiovascular disorder and cancers. Importantly, several human disorders associated with chaperones and proteases of the mitochondrial quality control system have been described. The mitochondrial proteome of mammals comprises about 1500 proteins (8). The vast majority of these (about 99%) are nuclear encoded, synthesized in the cytosol and imported posttranslationally through the TOM complex (figure 2 adapted from 9), which functions as the general mitochondrial import pore.
Following translocation across the mitochondrial outer membrane, proteins are imported into the matrix via the TIM complex. Mitochondrial proteins are typically imported as a linear polypeptide. This means that newly imported proteins are folded into their native conformation at their final destination often with the help of chaperones. The matrix contains about 500 proteins and within the matrix, the folding of newly imported proteins is assisted by the Hsp70 and chaperonin (Hsp60-Hsp10) complex (8). A recent proteomic study showed that nearly 50% of the matrix proteins interact with the Hsp60-Hsp10 system (10). This interactome includes a wide variety of matrix proteins that function in many metabolic pathways, including the respiratory chain, the citric acid cycle and fatty acid oxidation. Physiologically, Hsp60 and Hsp70 play a crucial role in maintaining a functional matrix proteome. When folding by these chaperones fails, the misfolded polypeptides are likely to aggregate and, in this case, they are either targeted for degradation or stabilized by Hsp78. This chaperone will disaggregate the polypeptides and subsequently assist Hsp70 in their refolding (figure 2).
Biological and structural features of the human Hsp60 chaperonin
Chaperonins are crucial for the proper functioning of bacterial and eukaryotic cells owing to their essential role in the folding of newly synthesized proteins as well as that of imported and stress-denatured proteins. Structurally, they form large barrel-shaped complexes made up of two stacked rings and a molecular mass of about 900 kDa. Substrate proteins with a mass of ~60 kDa are enclosed within the interior of the barrel for folding. Thus, the chaperonin complex can be viewed as a nanocage for the folding of individual substrate proteins (2,3). Chaperonins are commonly divided into two groups. Group I chaperonins are found in bacteria, mitochondria and chloroplasts and form a ring made up of seven subunits. Functionally, they cooperate with cochaperones that form the lid of the barrel. The bacterial system comprises GroEL and its cochaperone GroES, whereas the eukaryotic system is made up of Hsp60 and Hsp10. Group II chaperonins are present in archaea (thermosome) and the eukaryotic cytosol (TRiC). They usually form a ring that comprises eight subunits and function independent of cochaperones (3,11).
Human Hsp60 and its cochaperone Hsp10 are encoded by the HSPD1 and HSPE genes as proteins of ~61 and ~11 kDa, respectively. These are expressed at high levels in all tissues and are posttranslationally imported into the mitochondrial matrix. Moreover, defects in the human Hsp60-Hsp10 chaperonin system are the underlying cause of a subset of genetic neurodegenerative disorders, which is in line with the notion that different neurodegenerative disorders are linked to mutations in components of the mitochondrial quality control system. Studies with a temperature-sensitive yeast mutant provided the first evidence that Hsp60 functions as a folding factor that is essential in the biogenesis of mitochondrial proteins (12). Subsequently, a direct role of Hsp60 as an ATP-dependent chaperone was shown with fungal Hsp60 (13). It was moreover established that Hsp10 is a key component of the yeast Hsp60-Hsp10 system (14), which is essential for eukaryotic cell viability (15). In addition to its role as mitochondrial chaperonin, it also participates in processes outside of the mitochondrion such as inflammation, apoptosis and carcinogenesis. Similar to the bacterial chaperonin GroEL/GroES, Hsp60 adopts a cylindrical barrel formed by two stacked rings, containing a protein folding chamber that can be closed by lids made up of Hsp10 rings. This assembly allows repeated cycles of binding, encapsulation, folding and release of substrate proteins in an ATP-dependent fashion. Specifically, these cycles comprise conformational changes, driven by ATP binding and hydrolysis, allosteric communication between the Hsp60 rings as well as closing and opening of the folding chamber by the Hsp10 lid (3).
Structures of the human chaperonin complex
Despite the detailed mechanistic understanding of the bacterial chaperonin system, the human Hsp60-Hsp10 system is still poorly understood. However, recently detailed structures of the human chaperonin system were reported, providing profound molecular insight into the catalytic cycle of this complex. Figure 3 shows the structure of the human mitochondrial chaperonin complex that was obtained by protein crystallography at 3.15 Å resolution (7) in surface (left top panel) and ribbon (center top panel) representation. Overall, the complex is football-shaped and is made up of two Hsp60 rings with seven subunits, which are closed by a Hsp10 ring at each end that also contain seven subunits. Within the chaperonin rings, all Hsp60 subunits contain ADP. The double ring architecture of chaperonin complex is somewhat unexpected because it is thought to function as a single ring assembly, which are observed in the absence of nucleotides. Owing to its symmetrical shape, the top half of the complex is referred to as the north pole, while the bottom half is designated south pole. A top view of the complex (north pole) is provided in the top right panel and a bottom view (south pole) is presented in the bottom right panel. The structure of an individual Hsp60 subunit is shown in left bottom panel with ADP in blue ball and sticks.
. Hsp60 shares about 51% identity at the primary sequence level with bacterial GroEL and it contains a similar domain organization with an: (i) equatorial domain that is mainly composed of α helices and contains the nucleotide-binding site, (ii) an apical domain that comprises two β sheets and five α helices, and (iii) an intermediate domain that connects the equatorial domain and apical domain. The center bottom panel shows the structure of a single Hsp10 subunit that displays 31% sequence identity to bacterial GroES. Structurally, Hsp10 comprises a β-barrel structure made up of seven β-strands as well as a prominent mobile loop. The Hsp10 ring is bound to the Hsp60 ring via an interaction between the mobile loop of Hsp10 and the apical domain of Hsp60, respectively. The finding that all Hsp60 subunits of both rings contain ADP is intriguing because this symmetry is not observed in GroES; rather, both GroES rings contain either ATP or one ring is occupied by ATP, while the other has ADP. Therefore, the nucleotide symmetry observed in Hsp60 suggests that it employs a catalytic mechanism that is different from that of GroEL because it does not involve negative cooperativity between the rings.
It has been established that chaperonin oligomerization and double ring formation are ATP-dependent because in the absence of ATP single ring assemblies are observed (half-football), while in the presence of ATP double ring complexes (football) is found. To obtain insight into the nucleotide-dependency of Hsp60 ring formation, structures of an ATP bound double ring mimic, an ADP bound double ring assembly and ADP bound single ring complex were assessed recently (6). To obtain the structure of the ATP bound mimic, the Hsp60-Hsp10 complex was crystallized at 3.7 Å resolution in the presence of ATP and BeF3. Under these conditions, a stable football complex is generated upon ATP hydrolysis that is unable to progress through the catalytic cycle and fold substrate proteins. This structure represents the ground state and is presented in the left (surface representation) and center (ribbon representation) panel of figure 3, clearly revealing the double Hsp60 rings, containing 7 subunits each, that are capped at either end by tetrameric Hsp10 rings. All Hsp60 subunits contain ADP as indicated. Additionally, cryo-EM was employed to solve the structure of the half-football complex in the presence of ATP at 3.8 Å. A reconstruction of the half-football complex is shown in the right panel of figure 3 in ribbon representation with the single tetrameric Hsp60 and Hsp10 rings well resolved. All Hsp60 subunits contain ADP as indicated. In all structures, the Hsp60 subunits are in an extended conformation with its three domains (equatorial, intermediate and apical domain) clearly recognizable.
During the catalytic cycle of the bacterial chaperonin system, the GroEL subunits display significant conformational changes that are driven by ATP binding and hydrolysis. However, transition from the Hsp60-Hsp10 ground state to the ADP-bound football complex is not accompanied with conformational changes in the Hsp60 subunits. Nevertheless, significant structural changes occur in the Hsp10 subunits when the mitochondrial chaperonin complex progresses from the ground state towards the ADP-bound state. Specifically, this involves a rotation of the entire Hsp10 lid of the north pole (top of the complex) between seven- and fifteen-degrees following ATP hydrolysis as shown in figure 5 (PDB 6HT7: ground state mimic, PDB 6MRC: ADP-bound football).
Within the ground state football, the two Hsp60 rings are joined together via two contact sites that involve: (i) an interaction between Ser464 residues of opposing subunits as well as a hydrogen bond with Glu462, and (ii) a salt bridge between Lys109 and Glu105 of opposing subunits. These interactions are shown in the upper panel of figure 6. However, the two rings are joined together via one contact site within the ADP-bound football that only involve an interaction between Ser464 residues of opposing subunits as well as a hydrogen bond with Glu462 (bottom panel of figure 6). Thus, in the ground state chaperonin complex the two Hsp60 rings are connected through a hydrogen bond and a salt bridge, while only the hydrogen bond is present in the ADP-bound football. The importance of Ser464 was explored by replacing it either with cysteine or alanine. The cysteine mutant was unable to dissociate into single ring complex because the two Hsp60 rings were forced together via disulfide bonds, whereas the alanine variant could only form single ring complexes. The obtained double and single ring complexes were able to fold substrate proteins in vitro, although the double ring complex is probably more active.
Summarizing conclusion
It is well established that chaperones play an essential cellular role through inhibiting protein aggregation, resolving kinetically trapped intermediates and supporting protein folding. To this end, chaperones typically interact with substrate proteins to attain its biologically active conformation without being present in its final structure (2,3). In fact, chaperones are a key component of the cell’s quality control system that monitors the functional integrity of the proteome. It is therefore not surprising that defects of this quality control system are in humans associated with different pathologies, including neurodegenerative diseases, metabolic disorders and cancer (4). Each cell contains a set of unrelated molecular chaperones that operate in parallel, and often redundant, networks to ensure a functional proteome. Specifically, the following chaperone families are present in all domains of life: Hsp40, Hsp60, Hsp70, Hsp90, Hsp100 and the small Hsps (3). Members of the Hsp60 family are also known as chaperonins and represent multicomponent nanomachines that aid protein (re)folding in an ATP-dependent fashion. Chaperonins are divided into two groups. Group I chaperonins are found in bacteria, mitochondria and chloroplasts and form a ring made up of seven subunits. Functionally, they cooperate with cochaperones that form the lid of the barrel. The bacterial system comprises GroEL and its cochaperone GroES, whereas the eukaryotic system is made up of Hsp60 and Hsp10. Group II chaperonins are present in archaea (thermosome) and the eukaryotic cytosol (TRiC). They usually form a ring that comprises eight subunits and function independent of cochaperones (3,11). The human Hsp60-Hsp10 complex is localized to the mitochondrial matrix and plays a crucial role in the folding of newly imported proteins as well as the refolding of misfolded ones. It was estimated recently that about 50% of the matrix proteins interact with the Hsp60-Hsp10 system, thereby emphasizing its importance in maintaining a functional matrix proteome (10).
Recently, detailed structures of the human mitochondrial Hsp60-Hsp10 complex were reported, revealing ring-shaped complexes comprising two heptameric Hsp60 rings capped at either end by a heptameric Hsp10 ring (football complex), or one heptameric Hsp60 ring with a heptameric Hsp10 lid (half football complex) (6,7). Based on these structures, a model for the chaperonin reaction cycle was proposed (figure 7 adapted from 6). Within the Hsp60-Hsp10 reaction cycle, active single and double ring chaperonin complexes coexist. Upon exchange of ADP for ATP, oligomerization and formation of the football complex is induced. In the absence of nucleotides or presence of ADP, the Hsp60 subunits assemble into single ring complexes. Conceivably, ATP shifts the equilibrium towards the double ring complex. This suggests that its primary role is induction of oligomerization instead of providing energy for protein folding as has been established for the bacterial GroEL/GroES system. The presence of active single and double ring complexes within the Hsp60-Hsp10 catalytic cycle also suggests that the human chaperonin complexes is split between the two rings (equatorial split). Furthermore, the finding that all Hsp60 subunits contain ADP points towards a mechanism that does not involve negative cooperativity between the rings because this would inhibit ATP hydrolysis. The Hsp60-Hsp10 system employs therefore a mechanism that is different from that of the bacterial chaperonin complex because its GroEL subunits don’t contain the same nucleotide within its two rings. Finally, after ATP hydrolysis, Hsp10 dissociates and the substrate protein is released. This is in line with the finding that Hsp60 binds to Hsp10 in the presence of ATP but not ADP.
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