Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Apr 5.
Published in final edited form as: Structure. 2016 Mar 17;24(4):537–546. doi: 10.1016/j.str.2016.02.006

Ring separation highlights the protein folding mechanism used by the phage EL encoded chaperonin

Sudheer K Molugu 1, Zacariah L Hildenbrand 1, David Gene Morgan 2, Michael B Sherman 3, Lilin He 4, Costa Georgopoulos 5, Natalia V Sernova 6, Lidia P Kurochkina 7, Vadim V Mesyanzhinov 7, Konstantin A Miroshnikov 7, Ricardo A Bernal 1,*
PMCID: PMC4823152  NIHMSID: NIHMS763183  PMID: 26996960

Summary

Chaperonins are ubiquitous, ATP dependent protein-folding molecular machines that are essential for all forms of life. Bacteriophage φEL encodes its own chaperonin to presumably fold exceedingly large viral proteins via profoundly different nucleotide-binding conformations. Our structural investigations indicate that ATP likely binds to both rings simultaneously and that a misfolded substrate acts as the trigger for ATP hydrolysis. More importantly, the φEL complex dissociates into two single rings resulting from an evolutionarily altered residue in the highly conserved ATP binding pocket. Conformational changes also more than double the volume of the single-ring internal chamber such that larger viral proteins are accommodated. This is illustrated by the fact that φEL is capable of folding β-galactosidase, a 116 kDa protein. Collectively, the architecture and protein-folding mechanism of the φEL chaperonin are significantly different from those observed in group I and II chaperonins.

Graphical abstract

graphic file with name nihms763183u1.jpg

Introduction

The process of protein folding is one of the most important and challenging research topics of contemporary biochemistry and in spite of its central role in life, it is one of the least understood biophysical processes. Protein folding into a native three-dimensional structure is influenced by a number of intracellular factors including biophysical phenomena induced by the polar solvent, molecular chaperones and by specialized macromolecular complexes termed chaperonins (Booth et al., 2008; Dobson, 2004). In fact, it is estimated that 10–30% of all cellular proteins require the assistance of a chaperonin to fold properly into their functional three-dimensional structure (Booth et al., 2008; Dobson, 2004; Horwich et al., 2006; Reissmann et al., 2007).

Chaperonins are categorized into either group I or group II based on the need for a co-chaperonin and subunit heterogeneity (Horwich et al., 2006; Reissmann et al., 2007). Group I chaperonins are homo-oligomers with 7 subunits in each of two rings arranged back to back and require a co-chaperonin (Horovitz and Willison, 2005; Horwich et al., 2006). This is the most intensely studied chaperonin group (e.g. E. coli groEL/ES) for which the general mechanism of chaperonin-mediated protein folding was first described (Horovitz and Willison, 2005; Horwich et al., 2006; Reissmann et al., 2007). The group II chaperonins, such as the thermosome, Mm-cpn, TF55, and TriC are homo or hetero-oligomers with 8 or 9 subunits per ring (Braig et al., 1994; Cong et al., 2012; Willison, 2011; Yebenes et al., 2011). The group II chaperonin mechanism is also similar to that of the groEL/ES system but does not require a co-chaperonin to close the ring (Braig et al., 1994; Schoehn et al., 2000). Instead, the apical domains are rearranged to form protrusions that extend to form an iris-like structure that closes the cavity entrance after the hydrolysis of ATP and therefore replaces the co-chaperonin in function (Balch et al., 2008; Zhang et al., 2011).

Here, we present a structural and biochemical investigation of a newly discovered φEL chaperonin encoded by the Pseudomonas phage EL. The φEL chaperonin is the first of only two chaperonin groEL orthologs that have been identified in a phage genome (phage OBP is the other) (Cornelissen et al., 2012; Hertveldt et al., 2005). The studies presented here shed light on the unusual behavior of the φEL chaperonin that is evolved to better suit the protein folding requirements of a phage.

Results

Sequencing of the bacteriophage EL genome revealed a putative gene product 146 (gp146) that showed limited similarity (25% sequence identity) to the bacterial groEL chaperonin sequence (Hertveldt et al., 2005). Moreover, none of the phage EL predicted open reading frames had sequence similarity to any co-chaperonins. The φEL chaperonin protein-folding activity had to be confirmed before proceeding to structure determination and functional analysis. This was first investigated in-vivo through the use of an E. coli strain (MGM100) whose groEL/groES operon is under the control of the inducible arabinose promoter and transformed with a plasmid carrying the φEL chaperonin gene under the control of lactose promotor (Nielsen et al., 1999). This enabled us to ask if the φEL chaperonin could replace the native groEL/ES bacterial chaperonin function in the absence of groEL/ES expression and whether the host groES is required by φEL chaperonin. The characterization of gp146 as a bona fide chaperonin was confirmed when the growth of MGM100 E. coli cells in the absence of arabinose (which blocks further synthesis of the host-encoded groEL/ES chaperonin) was restored upon expression of the φEL chaperonin protein with the IPTG inducer (Figure 1A).

Figure 1. Functional properties of the φEL chaperonin.

Figure 1

Open in a new tab

(A) Chaperonin function is restored to bacterial cells (MGM100) that have had the native chaperonin expression under the control of an arabinose promoter. The cells were transformed with a plasmid carrying the φEL chaperonin gene under the lac promoter control. Shown are the growth curves without the addition of arabinose but in the presence of 1 mM IPTG (red) and absence of IPTG (green). (B) ATPase activity of the φEL chaperonin in the presence and absence of denatured α-lactalbumin substrate. The assay measures released inorganic phosphate (Pi) as a result of ATP hydrolysis by the chaperonin. φEL chaperonin hydrolyzes ATP only after adding the denatured α-lactalbumin substrate.

To investigate the protein folding mechanism of this phage φEL chaperonin, the effects of nucleotide binding and hydrolysis on conformational changes that drive the folding of the encapsulated substrate were explored. The φEL chaperonin was over-expressed in BL21 (DE3) E. coli cells, purified to homogeneity and flash-frozen for cryo-EM structural analysis. Purified φEL chaperonin was then incubated with 2mM ATP, in the absence of substrate. ATP hydrolysis appears to be inhibited by the lack of substrate and serves as a natural way to inhibit progression along the rest of the protein folding pathway. It is only after the addition of substrate (Ca2+ depleted α-lactalbumin) that ATP hydrolysis by φEL was observed (Figure 1B).

Open (ATP) φEL Conformation

The ATP treated φEL chaperonin reconstruction revealed a remarkable amount of detail to approximately 6.8 Å resolution based on the Fourier shell correlation method (Figure S1A). The overall architecture turned out to be similar to that of groEL with some notable differences. The ATP bound conformation has D7 symmetry and is open at both ends. This φEL conformation is 164 Å along the long axis and 143 Å along the short axis (Figure 2A and Table S1). The internal protein folding chamber of each ring has approximate dimensions of 49 Å parallel to the 7-fold symmetry axis and 105 Å perpendicular to the same axis (Figure 2B). There is also an internal gap of 44 Å between the equatorial domains of the two rings (Figure 2B). The entrance to each of the symmetric internal protein folding chambers has a diameter of 73 Å while the hole at the bottom of each chamber at the equatorial domain region is only 42 Å in diameter (Figure 2C and 2D).

Figure 2. ATP bound open conformation of the φEL chaperonin.

Figure 2

Open in a new tab

(A) The side view of the ATP bound φEL chaperonin reconstruction colored with a radial gradient from blue to red away from the 7-fold symmetry axis. (B) A central slab of the ATP bound φEL chaperonin. The purple line indicates the position of the equatorial domain. (C) An end view of the φEL chaperonin down the 7-fold symmetry axis. (D) A 4nm thick slab perpendicular to the 7 fold symmetry axis reveals many alpha-helices of the φEL equatorial domain.

Fitting of homology model coordinates into the ATP-φEL reconstruction revealed a lack of correspondence with density primarily in the apical domain. This was alleviated when the coordinates were subjected to geometry-based conformational sampling with the program DireX (Figure S2) (Schroder et al., 2007). Although not equivalent to a high resolution structure with accurate atomic positions, this fitting provides a reasonable representation of predicted secondary structural elements and the location of the ATP binding pocket (Figure S2C).

Closed (ADP) φEL Conformation

Purified chaperonin was incubated with 2 mM ADP and in the absence of protein substrate in an effort to mimic its ATP hydrolyzed conformational state. A cryo-EM dataset was collected and processed in the same way as the φEL-ATP dataset except D7 symmetry failed and only C7 symmetry could be used. This resulted in an 11.7 Å resolution reconstruction, based on the Fourier shell correlation method (Figure S1A). The structure that emerged from ADP treatment has a radically different conformation where the cavity opening is substantially diminished and the two heptameric rings have dissociated. Surprisingly, the overall dimensions are only slightly smaller to the dimensions of the open double-ring ATP conformation with 158 Å parallel to the 7-fold symmetry axis and approximately 147 Å perpendicular to it (Figure 3A and Table S1). However, there is clearly an expansion of the complex where the internal cavity now measures 115 Å parallel to the 7-fold symmetry axis and approximately 98 Å perpendicular to the 7-fold axis of symmetry (Figure 3B). Furthermore, the apical domain has been rearranged from an opening of 73 Å in the open ATP-bound conformation to about 20 Å in the ADP-bound closed conformation (Figure 3C compared to 2C). The heptameric equatorial domain hole maintains a consistent size of approximately 42 Å in diameter when compared to the φEL-ATP conformation and therefore serves as an internal magnification standard (Figure 3D compared to 2D). The entire equatorial domain, however, drops away from the cavity center by approximately 60 Å (movement of 76° when compared to the position of the equatorial plate in the φEL-ATP conformation) to allow the formation of the larger protein folding chamber (Figure 3B compared to 2B, purple lines). The double-ring dissociation of the φEL chaperonin into single rings was also observed by negative stain TEM (Figure S3).

Figure 3. ADP bound closed single-ring conformation of the φEL chaperonin.

Figure 3

Open in a new tab

(A) A side view of the ADP bound single ring φEL chaperonin. A protruding equatorial domain is clearly visible in the lower third of the structure (below red region). (B) A cross-section of the φEL map reveals the expanded internal chamber. The purple line indicates the position of the equatorial domain relative to its position in the ATP bound conformation in Figure 2B. (C) The top view of the φEL chaperonin highlights the iris-like lid formation. (D) The bottom view of the φEL single ring shows a protruding equatorial domain (blue and green).

Apo (Nucleotide-Free) φEL Conformation

Since the nucleotide-binding site appears to control the conformational state of the chaperonin, the next logical step was to obtain a reconstruction with the nucleotide-binding site devoid of any bound nucleotide. The resulting 9 Å apo-reconstruction revealed that the two heptameric rings come back together to form a double-ring closed conformation (Figure 4A and S1A). This apo conformation is slightly larger with dimensions that are 190 Å parallel to the seven-fold axis of symmetry and 163 Å perpendicular to this axis (Figure 4A and Table S1). The internal cavity appears to have diminished in size to 73 Å in both directions (Figure 4B). The opening at each end is still clearly closed with a diameter of approximately 27 Å (Figure 4C). The reconstructions of the ADP and Apo conformations reveal that the co-chaperonin function is supplanted by a conformational rearrangement of the apical domain that effectively closes each ring.

Figure 4. Nucleotide-free closed double-ring conformation of the φEL chaperonin.

Figure 4

Open in a new tab

(A) The side view of the φEL chaperonin apo. (B) A cross-section of the φEL map illustrates how the internal protein folding chamber. The purple line indicates the position of the equatorial domain relative to its position in the ATP and ADP bound conformations in Figures 2B and 3B, respectively. (C) The end view of the φEL clearly illustrates the closed apical domain with a structure that is similar to the iris-like lid seen in the closed single ring reconstruction in Figure 3C. (D) A cross-section perpendicular to the 7-fold symmetry axis.

The equatorial domain that protrudes into the cavity from each subunit is now nearly perpendicular to the seven-fold axis of symmetry (Figure 4B). This equatorial domain appears to have moved by approximately 45 Å into the cavity from the position it had in the ADP single ring (Figure 4B compared to 3B, purple lines). This same region appears to move another 15 Å into the cavity upon ATP binding to the nucleotide binding site (Figure 2B purple line).

Ring Separation and Internal Cavity Expansion

The dissociation of two heptameric rings observed by cryo-EM was confirmed by multiple biophysical methods including dynamic light scattering, analytical centrifugation, analytical chromatography and small angle neutron scattering. In each case, the formation of the single ring conformation can be easily detected confirming the results of the cryo-EM reconstructions. A possible explanation for the ring separation and internal cavity expansion is that one or more bacteriophage proteins cannot fold on their own because they may be too large for the host chaperonin. To test this hypothesis, we evaluated the ability of φEL to refold denatured β-galactosidase, a 116 kDa substrate that cannot be folded by the groEL/ES chaperonin (Ayling and Baneyx, 1996). The refolding activity of the φEL chaperonin was confirmed in-vitro when the denatured β-galactosidase was refolded restoring its ability to cleave ONPG (ortho-Nitrophenyl-β-galactoside) (Figure 5). The volume corresponding to atomic positions (electron density) was calculated for all three conformational states at a contour level right above noise for each normalized map to determine if the electron density volumes correspond to a loss of density after ring separation. These relative electron density volumes match what would be expected for the double vs single ring conformational states (Table S-1).

Figure 5. φEL chaperonin refolding of an exceedingly large substrate.

Figure 5

Open in a new tab

The activity of native, denatured and refolded β-galactosidase was detected by an increase in ONPG hydrolysis. The loss of activity of the denatured β-galactosidase served as a negative control as there was no detectable ONPG hydrolysis, indicating that the β-galactosidase could not fold by itself. In the presence of φEL chaperonin, the denatured β-galactosidase was refolded to its native conformation. The groEL/ES chaperonin was unable to fold the denatured β-galactosidase. The positive control included native β-galactosidase and all of the components in the reaction mixture except the chaperonin.

Validation of Size Difference Seen in the φEL Conformational States

The conformational differences seen in the cryo-EM reconstructions induced by the presence or absence of nucleotide (ATP, ADP, and APO) are also detectable by dynamic light scattering (DLS), analytical ultracentrifugation, size-exclusion chromatography and Small Angle Neutron Scattering (SANS) techniques (Figure S4).

DLS readings show polydispersity indices of less than 0.1 and peak widths of less than 4 nm. Such purity provides evidence that the chaperonin exhibits conformational stability in the absence of substrate (Figure S4A). Furthermore, the van Holde-Weischet plot of the analytical centrifugation also confirms that ATP hydrolysis causes inter-ring dissociation shifting the weight-average s-value from 20S for φEL-ATP to 16.4S for φEL-ADP (Figure S4B). Size-exclusion chromatography revealed the ability of the φEL chaperonin to assume its various conformational states after appropriate nucleotide treatment, as indicated by multiple chromatographic peaks (Figure S4C). Each peak contains highly purified target protein but a different appearance by negative stain and cryo-electron microscopy. Small Angle Neutron Scattering (SANS) analyses were performed on the chaperonin in the presence of ATP or ADP. The ATP bound conformation is slightly larger in size when compared to the ADP bound conformation with a radius of gyration (Rg) for the ATP conformation is ~72.7Å and ~71.0Å for the ADP conformation (Figure S4D–F).

The cryo-EM reconstructions, DLS, analytical ultracentrifugation, size exclusion chromatography and SANS results are all in agreement in terms of differences in particle size relative to the ATP, ADP and APO conformations.

Bioinformatics Analysis of the φEL chaperonin

Sequencing of the bacteriophage EL genome revealed a putative gene product 146 (gp146) that showed similarity and a 25% sequence identity to only the bacterial groEL chaperonin sequences and none of the group II chaperonins (Hertveldt et al., 2005). Moreover, none of the phage EL predicted open reading frames had sequence similarity to any known co-chaperonins. A multiple sequence alignment, using the program MUSCLE, was executed between the phage chaperonins, the group I chaperonins, and the group II chaperonins to identify conserved residues in φEL. When the φEL chaperonin sequence was aligned to various groEL sequences, the residues involved in the binding of ATP and its cofactor (Mg+2) in groEL are conserved in φEL except the amino acid alanine at the 92nd position in groEL where it is replaced with T in φEL (Figure S5A Box 1) (24, 25). GroEL residues involved in substrate recognition (Y199, Y203, F204, L234, L237, L259, V263, and V264) are either conserved or replaced with similar hydrophobic residues in the φEL chaperonin suggesting that substrate recognition is similar to groEL chaperonin (Figure S5A, Box 3 and Box 4)(Brocchieri and Karlin, 2000).

Discussion

The three reconstructions of the φEL chaperonin reveal profoundly different conformational states depending on the nucleotide bound to the nucleotide binding site. Moreover, these three φEL conformational states reveal a unique protein folding mechanism with characteristics that do not clearly fit into group I or group II chaperonins. This is due to unique features such as the dissociation of the double-ring structure into closed single rings upon ATP hydrolysis and the expansion of the internal chamber that allows the encapsulation and refolding of a much larger substrate protein.

Structural comparison of φEL with group I chaperonins

The φEL chaperonin is similar to group I chaperonins in that it is tetradecameric with seven fold rotational symmetry (Braig et al., 1994; Horwich et al., 2006). The similarity to group I members, however, ends there as there are significant differences (Table S2). First, the group I chaperonin groEL requires groES to close the protein-folding chamber while the apical domains of φEL rearrange and close the chamber after ATP hydrolysis. Second, inter-ring contacts between the φEL chaperonin subunits are in-register (1:1), where one subunit interacts with only one subunit directly across the inter-ring interface (Figure S5B). In groEL however, the inter-ring contacts are aligned in a staggered configuration (1:2), where one subunit interacts with two subunits across the inter-ring interface (Figure S5C) (Ranson et al., 2001; Saibil et al., 2013). Finally, negative inter-ring allostery in groEL restricts ATP binding to both rings and imposes an overall C7 symmetry with the cis-ring having bound ATP while the trans-ring is devoid of ATP (Ranson et al., 2001; Saibil, 2013; Saibil et al., 2013). The φEL-ATP reconstruction suggests that ATP binds to all subunits resulting in the observed D7 symmetry where both rings are open. The residues that control inter-ring contacts in groEL are mutated in the φEL chaperonin (Figure S5A Box 2) (Brocchieri and Karlin, 2000).

Structural comparison of φEL with group II chaperonins

The similarities between the φEL and group II chaperonins include ring closure via apical domain rearrangements that preclude the need for a co-chaperonin and an in-register subunit (1:1) alignment at the inter-ring interface (Table S2, Figure S5D) (Booth et al., 2008; Horovitz and Willison, 2005; Reissmann et al., 2007; Willison, 2011). However, the φEL chaperonin differs from the group II members in that it can facilitate the folding of non-specific substrates both in-vitro and in-vivo (Figs. 1A, 5), while the group II chaperonins are substrate specific (Booth et al., 2008; Horovitz and Willison, 2005; Reissmann et al., 2007; Willison, 2011). Furthermore, ATP hydrolysis in φEL induces conformational changes in the equatorial domain that triggers double-ring dissociation into closed single rings, while group II chaperonins do not dissociate into single rings. Lastly, group II chaperonins are homo or hetero-oligomers with at least 18 subunits while φEL is a homo-tetradecamer (Booth et al., 2008).

Structural similarities between φEL with other chaperonins

Chaperonin dissociation into single rings has been previously reported with the mitochondrial hsp60/10 chaperonin and in groEL mutants that dissociate into single rings (Chen et al., 2006; Nielsen et al., 1999; Nisemblat et al., 2015). It has been previously reported that the human mitochondrial chaperonin exists in dynamic equilibrium between single and double rings (Nisemblat et al., 2015). Bioinformatics analysis suggests that the ring dissociation in the mitochondrial chaperonin is likely due to mutations in the residues that are associated with inter-ring salt bridge contacts. These residues are responsible for negative allostery in groEL and are found mutated in the human mitochondrial hsp60 (Brocchieri and Karlin, 2000; Nielsen et al., 1999). A similar situation has been observed in the φEL sequence. The equivalent inter-ring salt bridge contact residues in φEL are also mutated resulting in the ability to form single ring intermediate (Figure S5A Box 2).

In groEL, point mutations such as A92T (located in the nucleotide binding site) and D398A (an important residue involved in groEL ATPase activity) trigger dissociation of the double-ring complex into single rings (Figure S5A Box 1 and 2) (Chen et al., 2006; Hartl et al., 2011; Kovacs et al., 2010). An equivalent A92T alteration was found in the φEL sequence which explains the ability of φEL to form a single-ring ADP intermediate. To test this hypothesis, we created a φEL mutant by reverting to the wild type groEL residue at position 92 which should prevent ring dissociation. This T91A mutation in φEL abolished all protein folding activity and inhibited ring separation (data not shown). The structural studies on the groEL D398A single ring mutant show a considerable expansion in the internal cavity volume that could encapsulate a substrate protein of 86 kDa (normally substrates up to ~57 kDa) (Chen et al., 2006; Hartl et al., 2011; Kovacs et al., 2010). The wild type φEL chaperonin therefore resembles the groEL mutants in terms of ring separation and cavity expansion.

The cavity expansion behavior observed with φEL was confirmed by the ability of φEL to restore enzymatic activity to denatured β-galactosidase, a 116 kDa subunit tetramer protein that cannot be folded by groEL/ES (Figure 5) (Ayling and Baneyx, 1996). This is not only indicative of the non-specific protein-folding nature of the φEL chaperonin but it supports the observed expansion of the protein-folding chamber in the closed ADP conformation that allows the chaperonin to accommodate exceedingly large phage proteins that are not accommodated by the host chaperonin (Table S3).

Studies on another groEL mutant E461K (a highly conserved residue involved in inter-ring salt-bridge contacts) revealed multiple alternations in the chaperonin structure and function (Sewell et al., 2004). This point mutation actually switched the usual staggered (1:2) inter-ring subunit alignment in groEL to an in-register (1:1) alignment that switched the symmetry from C7 to D7. This switch in the inter-ring contacts is associated with the loss of negative co-operativity in ATP binding and hydrolysis. Residue E461 is therefore critical for the two-stroke mechanism that inhibits ATP binding to the trans-ring (Sewell et al., 2004). The alignment between the φEL and groEL sequences revealed that the φEL chaperonin has a lysine that is equivalent to the groEL E461K mutation and therefore supports the φEL ATP and apo reconstructions where the 1:1 inter-ring subunit alignment is observed. Furthermore, this mutation suggests co-operative binding of ATP which would then lead to the observed D7 symmetry (Figure S5A box 2).

The groEL mutants not only support the φEL studies reported here, but also provide an explanation for why the φEL reconstructions take on the observed conformations. The structural characteristics of the φEL chaperonin in the presence of ATP and ADP correlates well with those observed in the groEL mutants. This includes the in-register (1:1) inter-ring subunit alignment and double-ring dissociation into single rings. Furthermore, ring dissociation in the mitochondrial chaperonin can also be explained as a consequence of the mutated residues involved in the inter-ring salt bridge contacts(Brocchieri and Karlin, 2000).

A phylogenetic tree derived from a neighbor-joining method identifies the presence of three clearly defined clusters that include group I chaperonins, group II chaperonins and a new single ring forming chaperonin group (Figure S5E). This new single ring forming chaperonin group (proposed group III) would include the φEL chaperonin presented here in addition to the recently discovered phage OBP chaperonin and the human mitochondrial hsp60.

Bacteriophages that encode chaperonins or co-chaperonins

One of the key reasons for a phage to carry a chaperonin or a co-chaperonin is to facilitate proper folding of viral substrate proteins that cannot be folded by the native chaperonin(Clare et al., 2006). Phages such as T4 and RB49 encode gp31 and CocO, respective co-chaperonins that interact with host groEL (Klein and Georgopoulos, 2001). Although the sequence identity between gp31 and groES is almost negligible (less than 10%), groEL-gp31 complex was absolutely required for the proper folding of gp23 (major capsid protein assembly of the viral capsid) (Clare et al., 2006). A structural investigation of groEL-gp31 complex revealed a considerable expansion in the resulting chamber that could then encapsulate the large, 521 amino acid gp23 substrate protein. The Pseudomonas phages EL and OBP are unusual in that they encode their own chaperonin and not a co-chaperonin (Cornelissen et al., 2012; Hertveldt et al., 2005).

Proposed φEL chaperonin Mechanism

When taken as a whole, the three significantly different conformations that arise from the presence or absence of nucleotide reveal a protein folding pathway that is considerably different from that of groEL (Figure 6). The proposed mechanism follows the hydrolysis of nucleotide with the corresponding three-dimensional reconstructions that logically follow the path of a protein from a misfolded to native state. The ATP-bound reconstruction illustrates a chaperonin primed for substrate binding with the two chambers fully accessible and in a completely open conformation. Substrate binding is then the trigger for ATP hydrolysis. Following ATP hydrolysis and inorganic phosphate release, the chaperonin undergoes a conformational change that results in lid formation and chamber closure through apical domain rearrangement. The substrate is simultaneously encapsulated within the internal cavity of each ring as the internal cavity itself undergoes a massive expansion as a result of displacement of the equatorial domain downward by about 60 Å. This large equatorial domain movement not only more than doubles the volume of the cavity but also leads to ring separation. Now sequestered within a sheltered environment away from interference from the cytoplasmic milieu, the unfolded or misfolded large protein substrate is allowed to fold properly. The details of the internal chamber must await a crystal structure but it is anticipated that a similar mechanism of swapping out of hydrophobic for hydrophilic side chains as seen with groEL will be employed in the φEL chaperonin as well (Horwich et al., 2006). The next step in the φEL chaperonin protein folding cycle follows the release of hydrolyzed nucleotide from the nucleotide binding sites in the equatorial domains that results in an equatorial domain movement back towards the center of the internal cavity by approximately ~45 Å. This movement of the equatorial domain not only compresses the internal cavity back to a volume that is slightly less than that seen in the ATP open conformation but now allows the two rings to come back together. Binding of ATP to the empty nucleotide binding sites moves the equatorial domain another 15 Å into the cavity and promotes a large rearrangement of the apical domain that opens each of the rings. This equatorial movement may help expel the folded substrate from the now open conformation.

Figure 6. Proposed novel φEL chaperonin protein folding cycle.

Figure 6

Open in a new tab

ATP binds simultaneously to both the φEL rings allowing substrate binding to both rings. Upon substrate binding, ATP is hydrolyzed triggering multiple simultaneous events including an apical domain rearrangement that creates the lid that closes the chamber opening, an equatorial domain movement that doubles the volume of the internal chamber and the separation of the two rings. Release ADP from the nucleotide binding pocket allows the rings to reunite into a closed double-ring conformation. Binding of ATP to both rings opens the chamber to release the folded substrate and returns the chaperonin to the open double-ring conformation.

It is clear that phage EL requires a chaperonin capable of folding unusually large viral proteins that cannot be folded by the smaller host chaperonin protein folding chamber that also does not expand. Upon further analysis of the genome, there are a few structural proteins that are very large and therefore potential chaperonin substrates (Table S3). Our experiments have demonstrated that the nucleotide determines the conformational state of the chaperonin and that this state is stable in the absence of the substrate. The substrate on the other hand acts as the trigger that drives the complex from one conformational state to the next. In other words, in the absence of substrate, ATP cannot be hydrolyzed in the open conformation and ADP cannot be expelled from the nucleotide binding sites in the closed conformation.

The phage EL chaperonin conformational intermediates represented by the three cryo-EM reconstructions provide a glimpse into a one-stroke protein folding mechanism. This mechanism likely includes positive inter- and intra-ring allosteric interactions that allow for a ring separation that more than doubles the protein folding chamber volume. This chaperonin is similar to the mitochondrial chaperonin in that they have both double and single-ring intermediates that are physiologically relevant for the folding of protein substrates. The similarities to both group I and II members in conjunction with novel characteristics makes it difficult to classify the φEL chaperonin as either a group I or group II member.

Experimental Procedures

Chemicals and cells

Unless otherwise stated, all chemicals, antibiotics and growth media were purchased from Sigma–Aldrich (St. Louis, USA). SDS-PAGE molecular weight standards were from BIO-RAD (Hercules, USA). The E. coli BL21 (DE3) cells purchased from New England Biolabs (Massachusetts, USA). All solutions were prepared using ultrapure deionized water and analytical grade reagents.

φEL Chaperonin Purification

The plasmid carrying gene146 was transformed into BL21 (DE3) for overexpression. Protein expression was induced with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside) at 30°C for four hours. The cells were harvested by centrifugation and resuspended in lysis buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM EDTA, 1 mM PMSF, and 0.02% NaN3. Cells were lysed with hen egg white lysozyme and centrifuged at 12,000 x g. The φEL chaperonin was precipitated from the supernatant using saturated ammonium sulfate to a final concentration of 40% v/v. The precipitated protein was resuspended in chromatography buffer (50mM HEPES (pH 7.5), 5 mM MgCl2, 50mM NaCl, 5mM EDTA and 1 mM PMSF) and loaded onto a superose-6 size-exclusion column. Purity of the sample was determined using SDS-PAGE.

groEL/ES chaperonin purification

The E. coli native chaperonin (groEL/ES) was expressed and purified from a K12 strain of E. coli lacking lacZ gene that is resistant to kanamycin antibiotic (Datsenko and Wanner, 2000). The native groEL/ES system was stimulated to over express in the cells by heat shocking the naturally growing bacteria at 42°C as described previously (Khandekar et al., 1993). Briefly, the E. coli cells were grown in a 1 liter 2xTY media at 37°C until the O.D600 reached 0.6. The temperature was immediately raised to 42°C and the cells were allowed to grow for an additional 4 hours. The increase in temperature (heat shock) causes over-expression of groEL and groES proteins. The cells were then harvested and processed as stated above for φEL with the exception of a saturated ammonium sulfate final concentration of 60% v/v. The precipitated protein was resuspended in φEL chromatography buffer and loaded onto a superose-6 size-exclusion column. The fractions corresponding to groEL were pooled together and purified further by subjecting the sample to a mono-Q anion exchange column. Additionally, the fractions corresponding to groES were pooled together and purified further by subjecting the sample to a mono-S cation exchange column. The purity of the samples were determined using SDS-PAGE.

φEL Sample Preparation for cryo-EM

The φEL chaperonin was concentrated to 2.5 mg/ml and incubated with 2 mM ATP or 2 mM ADP for 5 minutes before being flash-frozen for cryo-EM analysis. The nucleotide-free (APO) conformation was prepared by the addition of 100 mM EDTA to chelate the Mg2+ co-factor and 0.5 M NaCl to extract the nucleotide from the binding pocket. The excess salt and EDTA were then removed with a desalting column equilibrated with 50 mM HEPES (pH 7.5), 150 mM NaCl and 5 mM EDTA. The protein was concentrated to 2.5 mg/ml and flash-frozen for EM analysis.

Cryo-Specimen Preparation and Image Collection

Each sample was applied to a holey carbon film on 200-mesh copper grids (Quantifoil R2/2) that were glow discharged for 30s. Excess protein solution was blotted with a Whatman #1 filter paper and rapidly plunged into liquid ethane. Data for the φEL-ATP specimen was collected on an FEI F20 microscope (MRC-LMB, Cambridge, UK) operated at 200 kV. Images were recorded on Kodak SO-163 film at a magnification of 50,000 and at 2.0–4.0 μm underfocus. The micrographs were digitized using the KZA scanner at the MRC-LMB, Cambridge, UK. The φEL-ADP specimen data were collected on a JEOL 3200FS microscope (Indiana University, Bloomington, IN) operated at 300 kV. Ice-embedded images were acquired at a magnification of 69,000 and at 1.0–4.0 μm underfocus using a Gatan UltraScan 4000 charge-coupled device (CCD) camera. The APO-φEL specimen data was collected on a JEOL 2200FS microscope (UTMB, Galveston, TX) operated at 200 kV. CCD images were collected between 0.5–2.5 μm underfocus and at a magnification of 76,142.

Image Processing and 3D Reconstruction

The pixel sizes calculated for the ATP, ADP and APO datasets were 2.4 Å, 2.17 Å and 1.97 Å, respectively. Particle selection was performed using the Signature and EMAN software packages (Chen and Grigorieff, 2007; Ludtke et al., 1999). From an initial 40,613 particles in the ATP dataset, 18,778 particles in the ADP dataset and 61,180 particles from the APO dataset, 18,851, 13,242, and 58,477 were used in the final reconstructions, respectively. The defocus for each of the digitized micrographs and CCD frames was calculated using the program CTFFIND 3.0 to assess drift and astigmatism (Mindell and Grigorieff, 2003). The EMAN program CTFIT was used for flipping the phases in each of the micrographs. D7 symmetry was used for the ATP and APO datasets and C7 symmetry was used for the ADP dataset. φEL particles were grouped and averaged to generate 200 reference-free class averages using e2refine2d.py program of the EMAN2 software package. About 10–15 best looking class (Pettersen et al., 2004)averages for each reconstruction were then manually selected and used in the STARTCSYM program to generate a preliminary low resolution three dimensional model of the φEL chaperonin. Algorithms from EMAN, EMAN2, RELION and CTFFIND3 were used for each reconstruction. The refinement process was iterated until no further improvement in resolution was observed according to the Fourier shell correlation (FSC). A homology model of the φEL chaperonin was generated for the purpose of fitting the coordinates in to the φEL-ATP reconstruction using the coordinates of the cis ADP-Thermus thermophilus groEL chaperonin (PDB 1WF4) using the CPHmodels-3.0 server. All figures for reconstructions were prepared using UCSF Chimera (Petersen et al., 2010).

φEL Sample Preparation for SANS

The purified chaperonin sample was buffer exchanged with deuterated chromatography buffer (10 mM HEPES pH 7.5, 100 mM NaCl) and concentrated to 5 mg/ml. Open and closed φEL conformations were induced by incubating 5 mg of purified protein with 10 mM MgCl2 and either 5 mM ATP or 5 mM ADP. The APO-φEL sample was prepared by NaCl-EDTA treatment (as mentioned in the φEL Sample preparation for cryo-EM) and then buffer exchanged into deuterated chromatography buffer and concentrated to 5 mg/ml.

SANS Data Collection

The SANS experiments were performed using the Bio-SANS (CG3) of the high-flux isotope reactor at the Oak Ridge National Laboratory. Samples were measured at room temperature in sealed, 1mm path length quartz cuvettes at concentrations of 1 mg/ml for ADP and ATP samples. The neutron wavelength was set to 6 Å with a wavelength spread, Δλ/λ of 0.14. Scattered neutrons were detected with a 1 × 1 m helium filled two-dimensional (2D) position-sensitive detector with 192 by 192 pixels. Two sample-to-detector distances, 1.7 m and 8 m, were used to cover a q range between qmin, 0.003 Å−1, and qmax, 0.16 Å−1, where q is the magnitude of the scattering vector and is equal to 4πsin(θ)/λ and 2θ is the scattering angle. The samples and corresponding buffers were collected for approximately 1h at each detector setting to obtain good counting statistics. The raw 2D data were corrected for the detector pixel response and dark current before being azimuthally averaged to produce the 1D scattered-intensity profile, I(q) versus q (He et al., 2010). The data were placed on an absolute scale (cm−1) through the use of calibrated standards (Wignall and Bates, 1987). The reduced 1D profiles from the two detector distances were merged using Igor Pro 6.1 (WaveMetrics, Lake Oswego, OR). The reduced and merged data were produced by subtracting the I(q) for the buffer from that of the sample and included a constant to account for the difference in incoherent scattering that arises from the difference in hydrogen content of the samples.

The data were analyzed according to the method of Guinier for the radius of gyration, Rg. The Rg is a model-independent parameter that provides a measure of the size of a particle. Guinier analysis utilizes a series expansion of I(q) to derive an approximate Gaussian form for the intensity. I(q)≈ I(0)exp(-q2Rg2/3). A linear fit of ln(I(q)) versus q2 provides the intercept and slope, which are related to the zero-angle scattering intensity, I(0), and Rg, respectively. The expansion used to derive the Guinier approximation is applicable to only a limited range of low q values. For a compact particle, the approximation is valid for a value of q·Rg <1.3.

Dynamic Light Scattering

Dynamic light scattering (DLS) measurements were carried out using the Malvern Zetasizer nano instrument. Purified φEL chaperonin sample (0.05 mg/ml) in three different conformations were prepared as explained above (sample preparation for cryo-EM). A quartz cuvette was filled with 400 μl of each of the samples and the DLS measurements were taken at 25°C. The hydrodynamic radius of the chaperonin was measured every 10 seconds for an average of at least 100 measurements in each experiment for reliable statistics.

φEL Sequence Analysis

In order to find protein sequences that are related to φEL chaperonin, a Psi-Blast search was executed. Two iterations of the Psi-Blast search were performed to find chaperonin sequences with an e-value of 0.0, similarity greater that 25% and gaps no greater than 11%. The search identified only groEL chaperonin sequences. The top five hits from different organisms, mainly bacteria, were retrieved from the protein database, using the accession numbers provided by the Psi-Blast hits. The sequences were then aligned with φEL chaperonin using MUSCLE to identify the conserved regions in φEL that are also conserved in groEL chaperonins.

Phylogenetic Analysis

To investigate the evolutionary relationship between the phage chaperonins and the existing chaperonin groups, a multiple sequence alignment was performed between group I, group II and phage encoded chaperonins (Edgar, 2004). A variety of group I, group II chaperonins and the phage chaperonins were aligned using MUSCLE from NCBI. Poorly aligned regions were eliminated using the GBLOCKS server, making the alignment more suitable for phylogenetic analysis (Castresana, 2000). A neighbor-joining method was then employed on this alignment using MEGA5 software package to build a phylogenetic tree (Tamura et al., 2011). Default parameters provided by the MEGA5 software were used during phylogenetic tree construction.

MGM Viability and Growth Curve

The plasmid carrying gp146 (φEL chaperonin) and ampicillin resistance was transformed into MGM100 E. coli cells. The native groE gene of MGM100 was knocked out and supplanted with a plasmid carrying the groE gene with kanamycin resistance (Nielsen et al., 1999). The expression of the chaperonin is strictly regulated under the control of the arabinose inducer. The MGM100 cells containing the φEL plasmid as well as groE plasmid were grown at 37°C overnight in LB media containing 0.002% arabinose and diluted 1:1,000 into fresh LB media containing either 1 mM IPTG or no IPTG (as control). The native chaperonin expression was inhibited by withholding arabinose in the growth media while the φEL chaperonin expression was induced with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside) at 30°C for four hours.

Analytical Chromatography

The φEL chaperonin sample was prepared as described in the φEL sample preparation for cryo-EM section above to create the open, closed and apo conformations. Each of the conformations was then analyzed by size exclusion chromatography by loading 4.5 mg of protein onto a pre-equilibrated superose-6 column on an AKTA purifier 100 chromatography system (GE Healthcare Life Sciences). The sample was eluted with 2 column volumes of chromatography buffer.

ATPase Activity of φEL

Chaperonin activity results in ATP hydrolysis and release of inorganic phosphate (Pi) which can then be measure using the EnzChek Phosphate Assay kit (Molecular Probes, Leiden, Netherlands). In this assay, the purine nucleoside phosphorylase (PNPase) catalyzes a reaction where the released Pi reacts with the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) and converts it to a product ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine which has a maximum absorbance at 360nm (Webb, 1992). Purified φEL was diluted into the reaction buffer (100 mM Tris-HCl pH 7.5, 20 mM MgCl2, 2 mM NaN3) and incubated with 2 mM ATP to saturate the ATP binding pockets. The chaperonin was then incubated in a large excess of calcium depleted and reduced α-lactalbumin in the reaction buffer diluted with 0.2 mM dithiothreitol and 0.5 mM EDTA. The ATP hydrolysis by the φEL was then observed by the increase in the absorbance at 360 nm. The assay was repeated three times and the average of three experimental values were recorded.

Refolding of Guanidinium hydrochloride (GnHCl) denatured β-galactosidase

β-galactosidase was selected for the refolding assay because its activity can be measured spectrophotometrically using ortho-Nitrophenyl-β-galactoside (ONPG) which changes color upon hydrolysis by β-galactosidase. Native β-galactosidase was fully denatured by incubating 25ul of 20mg/ml with 25 ul of buffer containing 6 M guanidinium hydrochloride, 200 mM HEPES pH 7.5, 1mM DTT for 7 minutes. A 50 μM of the denatured β-galactosidase was diluted into 500 μl of reaction buffer containing 50mM HEPES pH 7.5, 5mM MgCl2, 50mM NaCl, 5mM ATP and 0.1 mg of ONPG to confirm that the enzyme was completely denatured and devoid of any enzymatic activity. This negative control reaction was left at room temperature overnight to ensure that β-galactosidase was unable to fold on its own. Two experimental reactions were performed to test the ability of the φEL and the groEL/ES chaperonin to utilize β-galactosidase as a substrate. The reactions were setup like the negative control but with the addition of 16 μM of either φEL or groEL/ES chaperonin. The positive control has 0.86 μM of native β-galactosidase without any chaperonin in the same reaction buffer used for the other reactions. The assays was allowed to proceed for 90 minutes while collecting 420nm absorbance readings every 10 seconds to monitor ONPG cleavage.

Analytical centrifugation measurements of φEL chaperonin

The analytical centrifugation experiments were performed using Beckman Optima analytical centrifuge. The φEL chaperonin sample was prepared as described in the φEL sample preparation for cryo-EM section above to create the open, closed and apo conformations. Each of the conformations was then analyzed by analytical chromatography experiment with a 24 hour run at 4°C. S-values were calculated from a van Holde-Weischet plot.

Supplementary Material

supplement

Highlights.

  1. The φEL chaperonin is the first of only two groEL orthologs encoded by a phage.

  2. ATP hydrolysis induces φEL chaperonin to dissociate into two closed single rings.

  3. The φEL chaperonin can fold substrate proteins without a co-chaperonin.

  4. The φEL chaperonin characteristics resemble both group I and II chaperonins.

Acknowledgments

The φEL reconstructions have been deposited in the EM databank (EMDB) with accession numbers EMD-6492 (ATP), EMD-6492 (ADP), and EMD-6492 (APO). This work was supported by NIH-NIGMS SC3GM113805, NSF-MRI 0923437 and Welch Foundation grant AH-1649 to Ricardo A. Bernal and Russian Fund for Basic Research grant #11-04-00935 to Lidia P. Kurochkina. We would like to thank Dr. Judy Ellzey and Dr. Peter Cooke, Director of the New Mexico State University EM facility for his help with negative stain TEM. The Bio-SANS of the Center for Structural Molecular Biology (FWP ERKP291) at Oak Ridge National Laboratory is supported by the Office of Biological and Environmental Research of the US Department of Energy. Research at the High Flux Isotope Reactor of Oak Ridge National Laboratory was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy.

Footnotes

Author Contributions:

S.M., R.B., D. M., M.B., performed cryo-EM data collection; S.M., purified protein samples and performed biochemical experiments; S.M., Z.H., R.B., prepared figures and wrote manuscript; L.H collected SANS data; C.G., N.S., L.K., V.M., K.M., sequenced and cloned φEL gene.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Ayling A, Baneyx F. Influence of the GroE molecular chaperone machine on the in vitro refolding of Escherichia coli beta-galactosidase. Protein science : a publication of the Protein Society. 1996;5:478–487. doi: 10.1002/pro.5560050309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science. 2008;319:916–919. doi: 10.1126/science.1141448. [DOI] [PubMed] [Google Scholar]
  3. Booth CR, Meyer AS, Cong Y, Topf M, Sali A, Ludtke SJ, Chiu W, Frydman J. Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT. Nat Struct Mol Biol. 2008;15:746–753. doi: 10.1038/nsmb.1436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braig K, Otwinowski Z, Hegde R, Boisvert DC, Joachimiak A, Horwich AL, Sigler PB. The Crystal-Structure of the Bacterial Chaperonin Groel at 2.8-Angstrom. Nature. 1994;371:578–586. doi: 10.1038/371578a0. [DOI] [PubMed] [Google Scholar]
  5. Brocchieri L, Karlin S. Conservation among HSP60 sequences in relation to structure, function, and evolution. Protein Sci. 2000;9:476–486. doi: 10.1110/ps.9.3.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular biology and evolution. 2000;17:540–552. doi: 10.1093/oxfordjournals.molbev.a026334. [DOI] [PubMed] [Google Scholar]
  7. Chen DH, Song JL, Chuang DT, Chiu W, Ludtke SJ. An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate. Structure. 2006;14:1711–1722. doi: 10.1016/j.str.2006.09.010. [DOI] [PubMed] [Google Scholar]
  8. Chen JZ, Grigorieff N. SIGNATURE: A single-particle selection system for molecular electron microscopy. Journal of Structural Biology. 2007;157:168–173. doi: 10.1016/j.jsb.2006.06.001. [DOI] [PubMed] [Google Scholar]
  9. Clare DK, Bakkes PJ, van Heerikhuizen H, van der Vies SM, Saibil HR. An expanded protein folding cage in the GroEL-gp31 complex. Journal of Molecular Biology. 2006;358:905–911. doi: 10.1016/j.jmb.2006.02.033. [DOI] [PubMed] [Google Scholar]
  10. Cong Y, Schroder GF, Meyer AS, Jakana J, Ma B, Dougherty MT, Schmid MF, Reissmann S, Levitt M, Ludtke SL, et al. Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle. The EMBO journal. 2012;31:720–730. doi: 10.1038/emboj.2011.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cornelissen A, Hardies SC, Shaburova OV, Krylov VN, Mattheus W, Kropinski AM, Lavigne R. Complete Genome Sequence of the Giant Virus OBP and Comparative Genome Analysis of the Diverse KZ-Related Phages. J Virol. 2012;86:1844–1852. doi: 10.1128/JVI.06330-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America. 2000;97:6640–6645. doi: 10.1073/pnas.120163297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dobson CM. Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol. 2004;15:3–16. doi: 10.1016/j.semcdb.2003.12.008. [DOI] [PubMed] [Google Scholar]
  14. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. doi: 10.1093/nar/gkh340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartl FU, Bracher A, Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis. Nature. 2011;475:324–332. doi: 10.1038/nature10317. [DOI] [PubMed] [Google Scholar]
  16. He LL, Piper A, Meilleur F, Myles DAA, Hernandez R, Brown DT, Heller WT. The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering. Journal of Virology. 2010;84:5270–5276. doi: 10.1128/JVI.00044-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hertveldt K, Lavigne R, Pleteneva E, Sernova N, Kurochkina L, Korchevskii R, Robben J, Mesyanzhinov V, Krylov VN, Volckaert G. Genome comparison of Pseudomonas aeruginosa large phages. J Mol Biol. 2005;354:536–545. doi: 10.1016/j.jmb.2005.08.075. [DOI] [PubMed] [Google Scholar]
  18. Horovitz A, Willison KR. Allosteric regulation of chaperonins. Curr Opin Struct Biol. 2005;15:646–651. doi: 10.1016/j.sbi.2005.10.001. [DOI] [PubMed] [Google Scholar]
  19. Horwich AL, Farr GW, Fenton WA. GroEL-GroES-mediated protein folding. Chem Rev. 2006;106:1917–1930. doi: 10.1021/cr040435v. [DOI] [PubMed] [Google Scholar]
  20. Khandekar SS, Bettencourt BM, Kelley KC, Recny MA. A simple and rapid method for the purification of GroEL, an Escherichia coli homolog of the heat shock protein 60 family of molecular chaperonins. Protein Expr Purif. 1993;4:580–584. doi: 10.1006/prep.1993.1076. [DOI] [PubMed] [Google Scholar]
  21. Klein G, Georgopoulos C. Identification of important amino acid residues that modulate binding of Escherichia coli GroEL to its various cochaperones. Genetics. 2001;158:507–517. doi: 10.1093/genetics/158.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kovacs E, Sun Z, Liu H, Scott DJ, Karsisiotis AI, Clarke AR, Burston SG, Lund PA. Characterisation of a GroEL single-ring mutant that supports growth of Escherichia coli and has GroES-dependent ATPase activity. J Mol Biol. 2010;396:1271–1283. doi: 10.1016/j.jmb.2009.11.074. [DOI] [PubMed] [Google Scholar]
  23. Ludtke SJ, Baldwin PR, Chiu W. EMAN: Semiautomated software for high-resolution single-particle reconstructions. Journal of Structural Biology. 1999;128:82–97. doi: 10.1006/jsbi.1999.4174. [DOI] [PubMed] [Google Scholar]
  24. Mindell JA, Grigorieff N. Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol. 2003;142:334–347. doi: 10.1016/s1047-8477(03)00069-8. [DOI] [PubMed] [Google Scholar]
  25. Nielsen KL, McLennan N, Masters M, Cowan NJ. A single-ring mitochondrial chaperonin (Hsp60-Hsp10) can substitute for GroEL-GroES in vivo. Journal of bacteriology. 1999;181:5871–5875. doi: 10.1128/jb.181.18.5871-5875.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nisemblat S, Yaniv O, Parnas A, Frolow F, Azem A. Crystal structure of the human mitochondrial chaperonin symmetrical football complex. Proceedings of the National Academy of Sciences of the United States of America. 2015;112:6044–6049. doi: 10.1073/pnas.1411718112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Petersen TN, Nielsen M, Lundegaard C, Lund O. CPHmodels-3.0-remote homology modeling using structure-guided sequence profiles. Nucleic Acids Research. 2010;38:W576–W581. doi: 10.1093/nar/gkq535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF chimera - A visualization system for exploratory research and analysis. Journal of Computational Chemistry. 2004;25:1605–1612. doi: 10.1002/jcc.20084. [DOI] [PubMed] [Google Scholar]
  29. Ranson NA, Farr GW, Roseman AM, Gowen B, Fenton WA, Horwich AL, Saibil HR. ATP-bound states of GroEL captured by cryo-electron microscopy. Cell. 2001;107:869–879. doi: 10.1016/s0092-8674(01)00617-1. [DOI] [PubMed] [Google Scholar]
  30. Reissmann S, Parnot C, Booth CR, Chiu W, Frydman J. Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins. Nat Struct Mol Biol. 2007;14:432–440. doi: 10.1038/nsmb1236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol. 2013;14:630–642. doi: 10.1038/nrm3658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Saibil HR, Fenton WA, Clare DK, Horwich AL. Structure and allostery of the chaperonin GroEL. J Mol Biol. 2013;425:1476–1487. doi: 10.1016/j.jmb.2012.11.028. [DOI] [PubMed] [Google Scholar]
  33. Schoehn G, Hayes M, Cliff M, Clarke AR, Saibil HR. Domain rotations between open, closed and bullet-shaped forms of the thermosome, an archaeal chaperonin. Journal of Molecular Biology. 2000;301:323–332. doi: 10.1006/jmbi.2000.3952. [DOI] [PubMed] [Google Scholar]
  34. Schroder GF, Brunger AT, Levitt M. Combining efficient conformational sampling with a deformable elastic network model facilitates structure refinement at low resolution. Structure. 2007;15:1630–1641. doi: 10.1016/j.str.2007.09.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sewell BT, Best RB, Chen S, Roseman AM, Farr GW, Horwich AL, Saibil HR. A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation. Nat Struct Mol Biol. 2004;11:1128–1133. doi: 10.1038/nsmb844. [DOI] [PubMed] [Google Scholar]
  36. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution. 2011;28:2731–2739. doi: 10.1093/molbev/msr121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Webb MR. A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proceedings of the National Academy of Sciences of the United States of America. 1992;89:4884–4887. doi: 10.1073/pnas.89.11.4884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wignall GD, Bates FS. Absolute Calibration of Small-Angle Neutron-Scattering Data. J Appl Crystallogr. 1987;20:28–40. [Google Scholar]
  39. Willison KR. Structural changes underlying allostery in group II chaperonins. Structure. 2011;19:754–755. doi: 10.1016/j.str.2011.05.008. [DOI] [PubMed] [Google Scholar]
  40. Yebenes H, Mesa P, Munoz IG, Montoya G, Valpuesta JM. Chaperonins: two rings for folding. Trends in biochemical sciences. 2011;36:424–432. doi: 10.1016/j.tibs.2011.05.003. [DOI] [PubMed] [Google Scholar]
  41. Zhang J, Ma B, DiMaio F, Douglas NR, Joachimiak LA, Baker D, Frydman J, Levitt M, Chiu W. Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure. Structure. 2011;19:633–639. doi: 10.1016/j.str.2011.03.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement
NIHMS763183-supplement.pdf (1.1MB, pdf)

RESOURCES