- Email: [email protected]
Chaperonins
159
Chaperonins Kerstin Braig Molecular chaperones are essential to all living organisms. Their key role consists of mediating protein folding within the cell. Recent functional studies have provided more detailed information about the function and regulation of the chaperone network. Highlights of the past year include the crystal structure determinations of the asymmetric G r o E L - G r o E S complex and of their isolated peptide-binding domains.
Chaperonins Chaperonins are found in all organisms and symbiotic organelles. They are large double ring structures consisting of 14, 16 or 18 subunits and mediate protein folding in Figure 1
Addresses Medical Reseach Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK; e-mail: [email protected] Current Opinion in Structural Biology 1998, 8:159-165 http://biomednet.com/elecref/0959440X00800159 ~, Current Biology Ltd ISSN 0959-440X Abbreviations CCT chaperonin containing TCP-1 DHFR dihydrofolate reductase hsp heat shock protein RBP Rubisco-binding protein TCP-1 t-complex polypeptide 1 TF55 thermophilic factor 55 EL
Introduction: the role of molecular chaperones in protein folding I,ittle is known about the mechanism by which newly synthesized proteins fold within the cell. In 1973, Anfinsen [1] showed that polypeptides can fold spontaneously under in e'itro conditions and reach their native state. Under nonideal conditions such as those that prevail in the cell, however, the folding of newly synthesized polypeptides is often inefficient as a result of competing off-pathway reactions. A group of specialized proteins, called molecular chaperones, has recently been identified as playing an essential role in enabling polypeptides to reach their biologically active form within a number of cellular corapartments [2,3°]. The term molecular chaperone describes members of several structurallv and genetically unrelated protein farailies that share the ability to recognize non-native conformations of other proteins and interact with them, without being a component of the final functional structure [4]. Two major families have been recognized: the DnaK, DnaJ, GrpE or heat shock protein (hsp) 70 family, and the chaperonin family (Table 1). Members of the hsp70 family interact with newly translated polypeptides both during translation and after their release from the ribosomes [2,5]. Their fundamental role is determined by their ability to bind to extended, hydrophobic peptide segments and to release them in an ATP-dependent manner in an unfolded conformation. Folding then occurs either spontaneously or with members of the chaperonin family. Chaperonins do not directly interact with newly synthesized polypeptides at the ribosome level. It has been suggested that the two chaperone systems form a lateral network of cooperating proteins ([6"',7°°]; Figure 1).
Aggregates
Native protein Current Opinion in Structural Biology
Model for cooperation between the hspT0 (DnaK) and chaperonin (GroEL) families in protein folding. The unfolded protein (U) is shown emerging from the ribosome (top) via several intermediates, that are either kinetically trapped (luc) or committed to fold (Ic) to the native state or to aggregates. Either GroEL (EL) or both DnaK (K) and Dna.I (J) bind to aggregation-prone folding intermediates in order to prevent aggregation. Binding of folding intermediates to DnaK and DnaJ lowers the concentration of free intermediates, thereby reducing the risk of a concentration-dependent off-pathway reactionl Intermediates dissociate from DnaK-DnaJ in a process controlled by ATP and GrpE (E} and then either rebind to DnaK-DnaJ or bind to GroEL and fold to the native state or aggregate. While there is genetic and biochemical evidence that, in the eukaryotic cytosol, hsp70 proteins interact with the nascent polypeptide chains [18,49], no co-translational interaction has been observed in the bacterial cytoplasm either with the hsp70 homologue DnaK or with GroEL [50], instead interaction has been observed with trigger factor (TF), a peptidyl prolyl isomerase [51,52]. Although there has been considerable argument favoring a pathway of successive interactions between hsp70 and the GroEL homologue hsp60 [3°,53,54], studies with deletion mutants have proven that DnaK is not an essential component of the protein folding pathway at all temperatures (whereas GroEL is) and that a sequential interaction cannot be essential [55].
160
Macromolecular assemblages
an ATP- dependent reaction. T h e y are generally divided into two groups ('IZable 1). T h e first group contains chaperonins originally found in the evolutionarily related compartments of the bacterial cytosol (GroEL), the mitochondrial matrix (hsp60) and the stroma of the chloroplasts (Rubisco-binding protein, RBP). T h e y are characterized by a high degree of sequence homology. In contrast, the second group shows a lower, but still significant, level of sequence homology. T h e group contains chaperonin containing TCP-1 (CCT), which is found in the eukaryotic cytosol [8], as well as archaebacterial chaperonins, the thermosome [9] and thermophilic factnr 55 (TF55) [10] (for a review, see [11]). While TFS5 [12] and the thermosome are composed of only two subunit species, the eubacterial chaperonin C C T contains tip to nine different subunit types [8,13,14]. So far, C C T is the only chaperonin identified in the eukaryotic cytosol. T h e striking difference between C C T and all other chaperonins is its very heteromeric compositinn: it comprises seven to nine [13,14] major polypeptide components, each of them independently encoded by highly diverged genes [12,14]. It has been suggested that
this genetic complexity indicates that C C T functions on a more elaborate level than the one- or two-subunit type chapemnins [11]. It has been shown that C C T facilitates the in s'itro folding of actin [15,16], tubulin [16,17], firefly luciferase [18] and Gc:transducin [7], and that it binds newly synthesized actin and tubulin [19]. It has also been suggested that C C T plays a role in the organization of the neuronal cytoskeleton [20]. GroEL is the best characterized chaperonin and therefore will be used in this review as a representative of chaperonins in general. It is a homo-oligomeric complex consisting of two heptameric rings composed of 57 kDa subunits that are stajcked back to back. On the basis of electron microscopy studies [21], it has been suggested that nonnative polypeptides are bound inside the central cavity that is enclosed by the subunits of each ring. q\vo groups [22",23"], using amide proton exchange studies, concluded that the polypeptides bound to GroEL are in a molten-globule-like state, meaning they are lacking in significant tertiary structure and, as a consequence, may have exposed hydrophobic regions while still containing native-like seconda U structure. In contrast, Goldberg eta/. [24"] found evidence that the bound polypeptide is in a metastable state
Table 1 Components of the hsp70 and chaperonin system. Bacteria
Eukaryotes
Compartment (in eukaryotes)
Properties
hsp70 system DnaK
hsp70, BiP
Cytosol, mitochondria, endoplasmic reticulum lumen
70 kDa protein with ATPase activity, binds extended peptides, interacts with hsp40 or its homologues and GrpE, required for post-translational protein import
DnaJ
hsp40
Cytosol, mitochondria, endoplasmic reticulum lumen
40 kDa protein, binds unfolded proteins, interacts with hsp70 or its homologues, stimulates their ATPase activity
GrpE
GrpE
Mitochondria
20 kDa protein, nucleotide exchange factor for hsp?0 or its homologues
hsp60-hspl 0
Mitochondria
Homo-oligomer, two rings of 760 kDa subunits, ATPase activity, binds folding intermediates, mediates protein folding together with co-chaperonin GroES (hspl 0)
RBP
Chloroplasts
GroEL homologue with two homologous subunit species, (z and [3, ATPase activity, interacts with cpn20 (the cofactor of cpn60) in folding of Rubisco subunits
Chaperonin system Group I GroEL-GroES
Group II TF55
Hetero-oligomer, two rings of 9 x 55 kDa subunits, two subunit species with homology to CCT, ATPase activity, binds folding intermediates, promotes folding in Archaea Hetero-oligomer, two rings of 8 x 55 kDa subunits, two subunit species homologous to TF55, ATPase activity
Thermosome CCT
Cytosol, nucleus
Hetero-oligomer, two rings of 8 or 9 x 55 kDa subunits, 7-9 different subunits, ATPase activity, binds folding intermediates, promotes folding and possibly assembly of cytosolic proteins
Chaperonins Braig
and that a native-like global topology is still present. These and many other results imply that GroEL is able to interact with a protein at several distinct steps during the folding process - - ranging from early intermediates lacking intrinsic stability to late-folding intermediates with native-like stability. While early kinetically trapped intermediates may be unfolded upon interaction with GroEL, the conformation of late intermediates is not affected, due to a high degree of intrinsic stabilization [25]. Ahhough ATP hydrolysis alone is sufficient to induce the productive release of substratcs that subsequently efficiently fold in the absence of chaperonins, many substrates require interaction with the co-chaperonin GroES, a homooligomeric single ring protein consisting of seven 10 kDa subunits (Figure 2).
161
GroEL-GroES structure T h e 'native' unligandcd GroEL structure is cylindrical with an overall diameter of 135 A and a height of 145 A, enclosing a central channel 45 A in diameter [26]. Each subunit is composed of 547 residues that fold into three flmctionally distinct domains: the highly ordered, mostly o~-helical equatorial domain; a small intermediate domain; and the highly flexible apical domain.
Equatorial domain T h e equatorial domains (one in each ring) form the structural base for the entire complex, providing most of the contact sites between subunits, both within each ring and across the equatorial plane. It is the largest domain and contains both the resolvable parts of the N-terminal and the C-terminal regions of the polypeptide chain. The 24 C-terminal
Figure 2
GroES
~
~
%
~
~
U
~ ' ~ U
or v
TRA.s
#
U or luc
Current Opinion in Structural Biology
Scheme for a reaction cycle of GroEL-mediated protein folding. An asymmetric GroEL-GroES complex with a high-affinity substrate-binding site (indicated by unshaded intermediate (I) domains) binds ATP and either unfolded polypeptides (U) or kinetically trapped folding intermediates (luc). Even though the binding reaction is diffusion-controlled, it is about ten times faster [56 °] than the reaction leading to the low-affinity state (indicated by dark grey I domains). Since the conformational switch to the state with low substrate affinity is a relatively slow reaction, substrate binding is very likely at cellular concentrations. After ATP is bound to the asymmetric GroEL-GroES complex, another GroES is bound, forming a symmetrical football-shaped complex. The binding of the second GroES leads to large hinge movements of the apical (A) domains (Figure 3) into the cis position. As a result of these movements, the surface of the cis cavity assumes a polar character [35"',37"]. This in turn initiates the folding reaction by displacing the substrate protein into the lumen of the cavity. Alternatively, an unproductive release of the bound polypeptide from the trans position into the bulk solution could occur. Hydrolysis of ATP in the cis ring discharges GroES and ADP from the trans ring and simultaneously restores an asymmetric complex with high affinity for ATP in the trans ring. Hydrolysis of ATP in the trans ring leads to the release of GroES and the folding intermediate from the cis ring. The released polypeptide is either native (N), committed to fold (Ic), or kinetically trapped. The light grey region indicates the part of the equatorial domain with a high degree of disorder. DP, ADP; E, equatorial domain; TP, ATP.
162 Mecromolecularassemblages
residues are apparently disordered in all custal structures and remain unresolved, which is consistent with the presence of four GGM repeats in the equatorial domain's sequence. Electron microscopy reconstructions [27] and small-angle neutron scattering studies [28] showed additional density in the central channel, on the level of the equatorial domains, which might account for these residues. The nucleotide-binding site of each subunit is located at the top of the equatorial domain, facing the central channel. It contains a set of conserved loops including the G D G T T sequence, a motif that is highly conserved among all chaperonins. The crystal structure of GroEL with bound ATPyS revealed that this sequence is a phosphate-binding loop that coordinates with all the phosphate oxygens either directly or indirectly via magnesium ions [29].
Apical domain The apical domain forms the other end of the G r o E b cylinder. It contains the binding sites both for substrate protein and co-chaperonin and is the least traceable part of the GroEL structure. The high atomic temperature factors in this area, combined with the observed differences between noncrystallographic symmetry-related molecules, suggest an intrinsic flexibility [30] that appears to be necessary for accommodating a wide range of substrate proteins on its hydrophobic surface. The st, bstrate proteins span a wide spectrum of molecular mass and chemico-physical properties [31,32"], defying a rigid lock-key model for interaction. T h e necessary degree of structural flexibility can be reached either globally, through rigid-body movements of the entire domain (for different sobstrate sizes), or locally on the channel surface, through mobile loops (for different protein conformations). It is assumed that the general substrate recognition and binding mechanism is based on hydrophobic interactions [33"].
Intermediate domain T h c smallest of the three domains, called the intermediate domain, seems to act mainly as a linker between the equatorial and the apical domains. Hinge-like connections to both domains on either side of the intermediate domain allow for the large rigid-body movements observed in electron microscopy reconstructions [34] and in a comparison of the crystal structure of the GroEL-GroES complex [35"] with the structure of GroEL alone. The main purpose of this domain seems to be the transmission of allosteric changes between the equatorial and apical domains. This hypothesis is further supported by the fact that salt bridges formed between the intermediate domain and the apical domain of adjacent subunits play a crucial role in the observed positive cooperativity of nucleotide binding and hydrolysis within one ring, and in the negative cooperativity between rings [36,37"',38]. Also, Fenton eta/. [39] observed that mutations in the intermediate domain affect all of the measurable functions of GroEL, which supports the view that the intermediate domain plays a major role in signal transmission.
GroES The co-chaperonin GroES forms a single ring structure and is composed of seven subunits each with a molecular weight of 10 kDa. The crystal structure of GroES alone shows a hollow dome-shaped structure with an outer diameter of 70-80 A, a height of 30 A, and an inner core 30]X in diameter and 20 A in height [40,41]. The central structural element in each subunit is a [3 barrel with two [8-hairpin loops: one reaching upwards to form the top of the dome, the other protruding from the base in order to interact with its chaperonin partner, GroEL. The loops involved in GroEL binding are flexible and could not be resolved in the crystal structure [40]. A comparison of the structure of the asymmetrical G r o E L - G r o E S complex with that of GroEL alone reveals two large rigid-body movements of entire domains [35"°]. Upon binding GroES, the apical domain of GroEL moves upwards out of the equatorial plane by 60 °, while twisting around by 90 °. This opening of the apical domains almost doubles the volume of the cavity inside GroEI,, which also becomes continuous with the dome of GroES. At the same time, the intermediate domain shifts by 25' towards the equatorial domain (Figure 3). T h e latter movement brings residue D398 of the intermediate domain as an additional ligand into hydrogen-bonding distance with the magnesium ion at the nuclentide-binding site. Mutational studies suggest that D398 is involved in ATP hydrolysis [42].
Reaction pathway of G r o E L - G r o E S m e d i a t e d folding In spite of the considerable research effort directed towards explaining the mechanism by which GruEL assists folding, little is known about the fate of a polypeptide undergoing a cycle of chaperonin-mediated protein folding. A basic model seems to be in place but interpretations sometimes vary to the point of giving rise to fierce debate. Based on the observation that polypeptides appear to be held within the central cavit'~; it has been suggested that GroEL performs its flmction by providing a so-called 'box of infinite dilution' [43] in which the substrate protein folds while awfiding unproductive interactions with other unfolded proteins. According to this model, release of the polypeptide would occur after it has reached its native state. In successflfl refolding experiments with isolated apical domains (residues 191-345), Zahn et a/. [22"] demonstrated that neither the presence of a central cavity nor the allosteric properties of the GroEL complex are essential for successful binding and refolding in vitro. They assume that the allosteric changes observed upon binding of GroES and ATP regulate the affinity of the complex for substrates in vivo. Jackson eta/. [44] proposed a model in which GroEL binds and unfolds aggregation-prone or kinetically trapped fold-
Chaperonins Braig
Figure 3
(a)
4o
4o
(b) GroES
i#
2°
2o
CurrentOpinionin StructuralBiology Schematic representation of the rigid-body movements in the GroEL complex upon GroES binding. (a) Before GroES binding, the interface between the two rings of the GroEL complex is slightly deformed. The subunits of the cis ring are tilted inwards by about 4 °, while the subunits of the trans ring tilt outwards by approximately 2 °. The complementary movement of the trans ring is necessary in order to preserve the inter-ring contacts in the equatorial plane [35"]. (b) Binding of GroES is accompanied by two major hinge movements (H t and H2). The intermediate (I) domains move down towards the equatorial (E) plane by approximately 25 ° thus closing the nucleotidebinding pockets. The apical (A) domains of the cis ring swing upwards by some 60 ° relative to the equatorial plane. At the same time they twist around their long axis by 90 ° [34]. This movement brings the apical domains in close proximity to the mobile loops on the underside of GroES that form the interface between GroEL and GroES [35"].
ing intermediates. Studies using a release-deficient mutant of GroEL [45] suggest that ATP binding promotes the release of non-native proteins into the bulk solution, where they either fold to the native state, or rebind as a kinetically trapped intermediate to another GroEL molecule and assume their original unfolded state [7°',46,47]. Recent results using either undiluted Xenopus egg lysates or macromolecular crowding agents to mimic the physicochemical properties of the prokaryotic cytosol contradict the release-rebind model [48"'].
Conclusions Even though the protein folding pathways of the two major chaperone systems involved in protein folding are globallx understood, there is much debate about single aspects of the process. Simple outlines seem to be in place and some of the complexities of the process of chaperonin-mediated protein folding have yielded to structural and biochemical investigations while others remain obscure. This is particularly true when it comes to identifying the nature of polypeptide binding and concomitant fi)lding, and explaining the active participation of the cavity in the cis position relative to GroES in directing productive folding within it.
163
Abundant data produced by the many groups working in the field seem to support different models. Upon closer inspection, it turns out that the interpretations depend to a large extent on the definitions and experimental procedures used to mimic the conditions in the living cell. The further we go in our understanding of chaperone-mediated protein folding the more careful we have to be in minute details and the more important it is to reach an eventual consensus. One of the main problems in applying a single model to describe the function of chaperonins for all substrate proteins is the great variety of potential substrates: they vary greatly in the tendency of their compact intermediates to aggregate, and also in their rate of spontaneous refolding. One should always keep in mind that most in vitro studies have been carried out Using only a few selected substrate proteins, namely dehydrofolate reductase, malate dehydrogenase and rhodanase. The generality of conclusions based on these experiments remains to be firmly established.
Acknowledgements T h e author thanks Eric de la Fortelle and C h a p i n A Rodriguez for their critical reading and discussion of the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ° of outstanding interest 1.
Anfinsen CB: Principles that govern the folding of protein chains. Science 1973, 181:223-257.
2.
Buchner J: Supervising the fold: functional principles of molecular chaperones. FASEB J 1996, 10:10-19.
3.
Hartl FU: Molecular chaperones in cellular protein folding. Nature 1996, 381:571-580. An excellent introduction to the field of molecular chaperones and assisted protein folding, with special emphasis on the hspT0 family and GroEL. 4.
Ellis RJ: Proteins as molecular chaperones. Nature 1987, 328:378379.
5.
Frydman J, Hohfeld J: Chaperones get in touch: the Hip-Hop connection. Trends Biochem Sci 1997, 22:87-92.
6.
Buchberger A, Schroder H, Hesterkamp T, Schonfeld HJ, Bukau B:
•.
Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding. J Mo! Biol 1996, 261:328-333.
Studies with denatured luciferase show that DnaK and GroEL compete for binding to non-native proteins; therefore, in this folding reaction, DnaK and GroEL do not necessarily act in succession by promoting earlier or later folding steps, but rather form a lateral network of proteins. 7.
Farr GW, Scharl EC, Schumacher RJ, Sondek S, Horwich AL:
•.
Chaperonin-mediated folding in the eukaryotic cytosol proceeds through rounds of release of native and nonnative forms. Cell
1997, 89:927-937. This study examines the fate of newly synthesized cytosolic proteins bound to CCT in reticulocyte lysate and Xenopus oocytes. In both cases, the production of the native protein is strongly inhibited by the introduction of a chaperonin trap, which is able to bind but not to release substrate protein. The overall mechanism of CCT-assisted protein folding resembles that of GroEL (see annotation [48"']). 8.
Lewis VA, Heynes GM, Zheng D, Sailbil H, Willison K: T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol. Nature 1992,358:249-252.
164
9.
Macromolecular assemblages
Phipps BM, Typke D, Hegerl R, Volker S, Hoffmann A, Stetter KO, Baumeister W: Structure of a molecular chaperone from a thermophUic archaebacterium. Nature 1993, 361:475-477.
26. Braig K, Otwinowski Z, Hedge R, Boisvert DC, Joachimiak A, Horwich AL, Sigler PB:.The crystal structure of the bacterial chaperonin GroEL at 2.8 A resolution. Nature 1994, 371:578-586.
10. TrentJD, Nimmesgern E, Wall J, Hartl FU, Horwich AL: A molecular chaperone from the thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature 1991, 354:490-493.
27. Chen S, Roseman AM, Hunter AS, Wood SP, Burston SG, Ranson NA, Clarke AR, Saibil HR: Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryoelectron microscopy. Nature 1994, 371:261-264.
11. Kubota H, Hynes G, Willison K: The chaperonin containing tcomplex polypeptide 1 (TCP-1) multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. Eur J Biochem 1995, 230:3-16.
28. Thiyagarajan P, Henderson SJ, Joachimiak A: Solution structures of GroEL and its complex with rhodanase from small-angle neutron scattering. Structure 1996, 4:?9-88.
12. Knapp S, Schmidt-Krey I, Herbert H, Bergman T, Jornvall H, Ladenstein R: The molecular chaperonin TF55 from the thermophiiic archeon Sulfolobus solfataricus. A biochemical and structural characterization. J Me~ Bio/1994, 242:397-407. 13. Rommelaere H, van Treys M, Gao Y, Melki R, Cowan NJ, Vandekerckhove J, Ampe C: The eukaryotic cytosolic chaperonin contains t-complex polypeptide and several related subunits. Proc Nat/Acad Sci USA 1993, 90:11975-11979. 14. Kubota H, Hynes G, Came A, Ashworth A, Willison K: Identification of six Tep-1 related genes encoding divergent subunits of the TCP-l-containing chaperonin. Curr Bio/1994, 4:89-99. 15. Gao Y, Thomas JO, Chow RL, Lee GH, Cowan NJ: A cytoplasmic chaperonin that catalyzes ~-actin folding. Cell 1992, 69:10431050. 16. Lewis SA, Tian G, Vainberg IE, Cowan NJ: Chaperonin-mediated folding of actin and tubulin. J Cell Biol 1996, 132:1-4. 17. YaffeMB, Farr GW, Miklos D, Horwich AL, Sternlicht ML, Sternlicht H: TCP1 complex is a molecular chaperone in tubulin biogenesis. Nature 1992, 358:245-258. 18. Frydman J, Nimmesgern E, Ohtsuka K, Hartl FU: Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 1994, 353:111-117. 19. Sternlicht H, Farr GW, Sternlicht ML, Driscoll JK, Willison K, Yaffe M: The t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo. Proc Natl Acad Sci USA 1993, 90:9422-9426. 20. Roobol A, Holmes FE, Hayes NVL, Baines A J, Carden AJ: Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells. J Cell Sci 1995, 108:1477-1488. 21. Braig K, Simon M, Furuya F, Hainfeld JF, Horwich AL: A polypeptide bound by the chaperonin groEL is localized within a central cavity. Proc Nat/Acad Sci 1993, 90:3978-3982. 22. Zahn R, Buckle AM, Perret S, Johnson CM, Corrales FJ, Golbik R, Fersht AR: Chaperonin activity and structure of monomeric polyeptide binding domains of GroEL. Proc Nat/Acad Sci USA 1996, 93:15024-15029. The crystal structure of a monomeric polypeptide that corresponds to the apical domain of GroEL shows a well-ordered structure with the same fold as that of native GroEL. The isolated domain functions as a minichaperone (see annotation [33"]). 23. GroB M, Robinson CV, Mayhew M, Hartl FU, Radford SE: Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling. Protein Sci 1996, 5:2506-2513. Structural analysis using hydrogen exchange labeling reveals that during several cycles of GroEL-assisted folding of dihydrofolate reductase the protein is partially folded rather than being completely unfolded upon binding to GroEL. 24. Goldberg MS, Zhang J, Sondek S, Matthews CR, Fox Re, Horwich °. AL: Native-like structure of a protein-folding intermediate bound by the chaperonin GroEL. Proc Natl Acad Sci USA 1997 94:10801085. The structure of DHFR bound to GroEL was analyzed using hydrogen-deuterium exchange and NMR spectroscopy. The data indicate that central structural elements found in the native protein are also present in the GroEL-bound form of the protein. Since these structural elements are derived from distant parts of the primary structure, the authors conclude that a native-like global topology is present in folding intermediates that are bound to GroEL. 25. Lille H, Buchner J: Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding. Proc Natl Acad Sci USA 1995, 92:8100-8104.
29. Boisvert DC, Wang J, Otwinowski Z, Horwich AL, Sigler PB: The 2.4/~, crystal structure of the bacterial chaperonin GroEL complex with ATPIsS.Nat Struct Biol 1996, 3:1 ?0-17?. 30. Braig K, Adams P, Brunger AT: Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution. Nat Struct Biol 1995, 2:1083-1093. 31. Ewalt KL, Hendrick JP, Houry WA, Hartl FU: In rive observation of polypeptide flux through the bacterial chaperonin system. Ceil 199?, 90:491-500. 32. LorimerGH: A quantitative assessment of the role of chaperonin proteins in protein folding in vivo. FASEB J 1996, 10:5-9. "What proportion of all the proteins of Escherichia coil reach their native states with the assistance of the chaperonins proteins, GroEL and GroES?" The author provides an easy to follow calculation to answer that question for a given E. coil strain under standard conditions. 33. Buckle AM, Zahn R, Fersht AR: A structural model for GroELpolypeptide recognition. Proc Nail Acad Sci USA 1997, 94:35713575. This paper presents the high resolution structure of an N-terminal-tagged polypeptide corresponding to the isolated apical domain of GroEL. In the structure, the N-terminal tag of one molecule is bound to the active region of a neighbouring molecule. The data presented here are used to reconstruct how a peptide can bind to the GroEL complex. 34. RosemanAM, Chen S, White H, Braig K, Saibil HR: The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell 1996, 87:241-251. 35. Xu Z, Horwich AL, Sigler PB: The crystal structure of the •. asymmetric GroEL-GroES-(ADP) 7 chaperonin compex. Nature 199'7, 388:741-750. This landmark paper presents the long awaited crystal structure of the asymmetrical (GroES)7-(GroEL)14 complex. The structure shows that there is a massive upward movement of the GroEL apical domains, which is accompanied by a twisting rigid-body movement around a hinge at the junction of the intermediate and apical domains. It further reveals that as a result of these movements, the surface properties of the central cavity in the cis cavity dramatically change from hydrophobic to polar. If one assumes that protein binding is due to hydrophobic interactions, GroEL would lose its binding properties once the cavity becomes polar. 36. Yifrach O, Horovitz A: Two lines af allosteric communication in the oligomeric chaperonin GroEL are revealed by the single mutation Arg196/Ala. J Mol Biol 1994, 234:397-401. 37. White HE, Chen S, Roseman AM, Yifrach O, Horovitz A, Saibil HR: •Structural basis of the allosteric changes in the GroEL mutant Arg 197 --) Ala. Nat Struct Biol 1997, 4:690-694. Cryo-electron microscopy has been used to generate three-dimensional reconstructions of a GroEL mutant with weaker intersubunit contacts, in different nucleotide-bound states. In this mutant, the domains are more free to move about the intermediate domain at all ATP concentrations. The study provides new insights into the nucleotide-dependent allosteric switching of GroEL. 38. Aharoni A, Horovitz A: Detection of changes in pairwise interactions during allosteric transitions: Coupling between local and global conformational changes in GroEL Proc Natl Acad Sci USA 199"7, 94:1698-1702. 39. Fenton WA, Kashi Y, Furtak K, Horwich AL: Residues in chapronin GroEL required for polypeptide binding and release. Nature 1994, 371:614-619. 40. Hunt JF, Weaver AJ, Landry SJ, Gierasch L, Deisenhofer J: The crystal structure of the GroES co-chaperonin a~ 2.8 A resolution. Nature 1996, 379:37-45.
Chaperonins Braig
165
41. Manda SC, Mehra V, Bloom BR, Hol WGJ: Structure of the heat shock protein chaperonin-10 of Mycobacterium leprae. Science 1996, 271:203-207.
49. Nelson RJ, Ziegelhoffer T, Nicolet C, Werner-Washburne M, Craig EA: The translation machinery and 70 kDa heat shock protein cooperate in protein synthesis. Ceil 1992, 71:97-105.
42. Rye HS, Burston SG, Fenton WA, Beechem JM, Xu Z, Sigler P, Horwich AL: Distinct action of cis and trans ATP within the double ring of the chaperonin GroEL Nature 1997, 388:792-798.
60. Reid BG, Flynn GC: GroEL binds to and unfolds rhodanase pos~ranslationally. J Biol Chem 1996, 271:7212-7217.
43. Ellis RJ: Opening and closing the Anfinsen cage. Curr Biol 1994, 4:633-635.
51. Hesterkamp T, Hauser S, Lutcke H, Bukau B: Escherichia colitrigger factor is a propyl isomerase that associates with nascent polypeptide chains. Proc Nat/Acad Sci USA 1996, 93:4437-4441.
44. JacksonGS, Staniforth RA, Halsall DJ, Atkinson T, Holbrook JJ, Clarke AR, Burston SG: Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: Implications for the mechanism of assisted protein folding. Biochemistry 1993, 32:2554-2563.
52. ValentQA, Kendall DA, High S, Kusters R, Oudega B, Luirink J: Early events in preprotein recognition in E. coil: interaction of SRP and trigger factor with nascent polypeptides. EMBO J 1995, 14:54945505.
45. Weissman JS, Kashi Y, Fenton WA, Horwich AL: GroEL mediated protein folding proceeds by multiple rounds of binding and release of non-native forms. Ceil 1994, 78:693-702. 46. Ranson NA, Dunster NJ, Burston SG, Clarke AR: Chaperonins can catalyse the reversal of early aggregation steps when a protein misfolds. J Mo/ Bio/1995, 250:581-586. 47
Burston SG, Weissman JS, Farr GW, Fenton WA, Horwich AL: Release of both native and non-native proteins from cis-only GroEL ternary complex. Nature 1996, 383:96-99.
48. Martin J, Hartl FU: The effect of macromolecular crowding in o. chaperonin-mediated protein folding. Proc Nat/Acad Sci USA 1997, 94:1107-1112. The point of this study was to investigate the fate of newly synthesized proteins in a dense cytosolic solution or in the presence of macromolecular crowding agents. Under these conditions, the transfer to a chaperonin trap, which is able to bind but not to release substrate protein, is prevented. At a twofold dilution, a significant increase in the rate of transfer is observed. The authors come to the conclusion that the release of non-native polypeptide is not an essential feature of the productive chaperonin mechanism. See annotation [7"'].
53. Langer T, Lu C, Echols H, Flanagan J, Hayer MK, Hartl FU: Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 1992, 356:683-689. 54. Gragerov A, Nudler E, Komissarova N, Gaitanaris GA, Gottesman ME, Nikiforov V: Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia co//. Proc Nat/Acad Sci USA 1992, 89:10341-10344. 55. Bukau B, Walker GC: Cellular defects caused by deletion of the Escherichia coil DnaK gene indicate roles for heat shock protein in normal metabolism. J Bacterio/1989, 171:2337-2346. 56. Spatter H, Buchner J: How GroES regulates binding of nonnative protein to GroEL. J Bio/Chern 1997, 272:14080-14086. This kinetic study suggests that the slow release of substrate protein from the unproductive trans position in the asymmetric GroEL-GroES complex favours the binding of a second GroES. The formation of a symmetric complex ensures that the substrate protein is sequestered in a position underneath GroES. According to this model, the intrinsic binding characteristics of the asymmetric trans complex determine the sequence of events during the reaction cycle.
Chaperonins Kerstin Braig Molecular chaperones are essential to all living organisms. Their key role consists of mediating protein folding within the cell. Recent functional studies have provided more detailed information about the function and regulation of the chaperone network. Highlights of the past year include the crystal structure determinations of the asymmetric G r o E L - G r o E S complex and of their isolated peptide-binding domains.
Chaperonins Chaperonins are found in all organisms and symbiotic organelles. They are large double ring structures consisting of 14, 16 or 18 subunits and mediate protein folding in Figure 1
Addresses Medical Reseach Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK; e-mail: [email protected] Current Opinion in Structural Biology 1998, 8:159-165 http://biomednet.com/elecref/0959440X00800159 ~, Current Biology Ltd ISSN 0959-440X Abbreviations CCT chaperonin containing TCP-1 DHFR dihydrofolate reductase hsp heat shock protein RBP Rubisco-binding protein TCP-1 t-complex polypeptide 1 TF55 thermophilic factor 55 EL
Introduction: the role of molecular chaperones in protein folding I,ittle is known about the mechanism by which newly synthesized proteins fold within the cell. In 1973, Anfinsen [1] showed that polypeptides can fold spontaneously under in e'itro conditions and reach their native state. Under nonideal conditions such as those that prevail in the cell, however, the folding of newly synthesized polypeptides is often inefficient as a result of competing off-pathway reactions. A group of specialized proteins, called molecular chaperones, has recently been identified as playing an essential role in enabling polypeptides to reach their biologically active form within a number of cellular corapartments [2,3°]. The term molecular chaperone describes members of several structurallv and genetically unrelated protein farailies that share the ability to recognize non-native conformations of other proteins and interact with them, without being a component of the final functional structure [4]. Two major families have been recognized: the DnaK, DnaJ, GrpE or heat shock protein (hsp) 70 family, and the chaperonin family (Table 1). Members of the hsp70 family interact with newly translated polypeptides both during translation and after their release from the ribosomes [2,5]. Their fundamental role is determined by their ability to bind to extended, hydrophobic peptide segments and to release them in an ATP-dependent manner in an unfolded conformation. Folding then occurs either spontaneously or with members of the chaperonin family. Chaperonins do not directly interact with newly synthesized polypeptides at the ribosome level. It has been suggested that the two chaperone systems form a lateral network of cooperating proteins ([6"',7°°]; Figure 1).
Aggregates
Native protein Current Opinion in Structural Biology
Model for cooperation between the hspT0 (DnaK) and chaperonin (GroEL) families in protein folding. The unfolded protein (U) is shown emerging from the ribosome (top) via several intermediates, that are either kinetically trapped (luc) or committed to fold (Ic) to the native state or to aggregates. Either GroEL (EL) or both DnaK (K) and Dna.I (J) bind to aggregation-prone folding intermediates in order to prevent aggregation. Binding of folding intermediates to DnaK and DnaJ lowers the concentration of free intermediates, thereby reducing the risk of a concentration-dependent off-pathway reactionl Intermediates dissociate from DnaK-DnaJ in a process controlled by ATP and GrpE (E} and then either rebind to DnaK-DnaJ or bind to GroEL and fold to the native state or aggregate. While there is genetic and biochemical evidence that, in the eukaryotic cytosol, hsp70 proteins interact with the nascent polypeptide chains [18,49], no co-translational interaction has been observed in the bacterial cytoplasm either with the hsp70 homologue DnaK or with GroEL [50], instead interaction has been observed with trigger factor (TF), a peptidyl prolyl isomerase [51,52]. Although there has been considerable argument favoring a pathway of successive interactions between hsp70 and the GroEL homologue hsp60 [3°,53,54], studies with deletion mutants have proven that DnaK is not an essential component of the protein folding pathway at all temperatures (whereas GroEL is) and that a sequential interaction cannot be essential [55].
160
Macromolecular assemblages
an ATP- dependent reaction. T h e y are generally divided into two groups ('IZable 1). T h e first group contains chaperonins originally found in the evolutionarily related compartments of the bacterial cytosol (GroEL), the mitochondrial matrix (hsp60) and the stroma of the chloroplasts (Rubisco-binding protein, RBP). T h e y are characterized by a high degree of sequence homology. In contrast, the second group shows a lower, but still significant, level of sequence homology. T h e group contains chaperonin containing TCP-1 (CCT), which is found in the eukaryotic cytosol [8], as well as archaebacterial chaperonins, the thermosome [9] and thermophilic factnr 55 (TF55) [10] (for a review, see [11]). While TFS5 [12] and the thermosome are composed of only two subunit species, the eubacterial chaperonin C C T contains tip to nine different subunit types [8,13,14]. So far, C C T is the only chaperonin identified in the eukaryotic cytosol. T h e striking difference between C C T and all other chaperonins is its very heteromeric compositinn: it comprises seven to nine [13,14] major polypeptide components, each of them independently encoded by highly diverged genes [12,14]. It has been suggested that
this genetic complexity indicates that C C T functions on a more elaborate level than the one- or two-subunit type chapemnins [11]. It has been shown that C C T facilitates the in s'itro folding of actin [15,16], tubulin [16,17], firefly luciferase [18] and Gc:transducin [7], and that it binds newly synthesized actin and tubulin [19]. It has also been suggested that C C T plays a role in the organization of the neuronal cytoskeleton [20]. GroEL is the best characterized chaperonin and therefore will be used in this review as a representative of chaperonins in general. It is a homo-oligomeric complex consisting of two heptameric rings composed of 57 kDa subunits that are stajcked back to back. On the basis of electron microscopy studies [21], it has been suggested that nonnative polypeptides are bound inside the central cavity that is enclosed by the subunits of each ring. q\vo groups [22",23"], using amide proton exchange studies, concluded that the polypeptides bound to GroEL are in a molten-globule-like state, meaning they are lacking in significant tertiary structure and, as a consequence, may have exposed hydrophobic regions while still containing native-like seconda U structure. In contrast, Goldberg eta/. [24"] found evidence that the bound polypeptide is in a metastable state
Table 1 Components of the hsp70 and chaperonin system. Bacteria
Eukaryotes
Compartment (in eukaryotes)
Properties
hsp70 system DnaK
hsp70, BiP
Cytosol, mitochondria, endoplasmic reticulum lumen
70 kDa protein with ATPase activity, binds extended peptides, interacts with hsp40 or its homologues and GrpE, required for post-translational protein import
DnaJ
hsp40
Cytosol, mitochondria, endoplasmic reticulum lumen
40 kDa protein, binds unfolded proteins, interacts with hsp70 or its homologues, stimulates their ATPase activity
GrpE
GrpE
Mitochondria
20 kDa protein, nucleotide exchange factor for hsp?0 or its homologues
hsp60-hspl 0
Mitochondria
Homo-oligomer, two rings of 760 kDa subunits, ATPase activity, binds folding intermediates, mediates protein folding together with co-chaperonin GroES (hspl 0)
RBP
Chloroplasts
GroEL homologue with two homologous subunit species, (z and [3, ATPase activity, interacts with cpn20 (the cofactor of cpn60) in folding of Rubisco subunits
Chaperonin system Group I GroEL-GroES
Group II TF55
Hetero-oligomer, two rings of 9 x 55 kDa subunits, two subunit species with homology to CCT, ATPase activity, binds folding intermediates, promotes folding in Archaea Hetero-oligomer, two rings of 8 x 55 kDa subunits, two subunit species homologous to TF55, ATPase activity
Thermosome CCT
Cytosol, nucleus
Hetero-oligomer, two rings of 8 or 9 x 55 kDa subunits, 7-9 different subunits, ATPase activity, binds folding intermediates, promotes folding and possibly assembly of cytosolic proteins
Chaperonins Braig
and that a native-like global topology is still present. These and many other results imply that GroEL is able to interact with a protein at several distinct steps during the folding process - - ranging from early intermediates lacking intrinsic stability to late-folding intermediates with native-like stability. While early kinetically trapped intermediates may be unfolded upon interaction with GroEL, the conformation of late intermediates is not affected, due to a high degree of intrinsic stabilization [25]. Ahhough ATP hydrolysis alone is sufficient to induce the productive release of substratcs that subsequently efficiently fold in the absence of chaperonins, many substrates require interaction with the co-chaperonin GroES, a homooligomeric single ring protein consisting of seven 10 kDa subunits (Figure 2).
161
GroEL-GroES structure T h e 'native' unligandcd GroEL structure is cylindrical with an overall diameter of 135 A and a height of 145 A, enclosing a central channel 45 A in diameter [26]. Each subunit is composed of 547 residues that fold into three flmctionally distinct domains: the highly ordered, mostly o~-helical equatorial domain; a small intermediate domain; and the highly flexible apical domain.
Equatorial domain T h e equatorial domains (one in each ring) form the structural base for the entire complex, providing most of the contact sites between subunits, both within each ring and across the equatorial plane. It is the largest domain and contains both the resolvable parts of the N-terminal and the C-terminal regions of the polypeptide chain. The 24 C-terminal
Figure 2
GroES
~
~
%
~
~
U
~ ' ~ U
or v
TRA.s
#
U or luc
Current Opinion in Structural Biology
Scheme for a reaction cycle of GroEL-mediated protein folding. An asymmetric GroEL-GroES complex with a high-affinity substrate-binding site (indicated by unshaded intermediate (I) domains) binds ATP and either unfolded polypeptides (U) or kinetically trapped folding intermediates (luc). Even though the binding reaction is diffusion-controlled, it is about ten times faster [56 °] than the reaction leading to the low-affinity state (indicated by dark grey I domains). Since the conformational switch to the state with low substrate affinity is a relatively slow reaction, substrate binding is very likely at cellular concentrations. After ATP is bound to the asymmetric GroEL-GroES complex, another GroES is bound, forming a symmetrical football-shaped complex. The binding of the second GroES leads to large hinge movements of the apical (A) domains (Figure 3) into the cis position. As a result of these movements, the surface of the cis cavity assumes a polar character [35"',37"]. This in turn initiates the folding reaction by displacing the substrate protein into the lumen of the cavity. Alternatively, an unproductive release of the bound polypeptide from the trans position into the bulk solution could occur. Hydrolysis of ATP in the cis ring discharges GroES and ADP from the trans ring and simultaneously restores an asymmetric complex with high affinity for ATP in the trans ring. Hydrolysis of ATP in the trans ring leads to the release of GroES and the folding intermediate from the cis ring. The released polypeptide is either native (N), committed to fold (Ic), or kinetically trapped. The light grey region indicates the part of the equatorial domain with a high degree of disorder. DP, ADP; E, equatorial domain; TP, ATP.
162 Mecromolecularassemblages
residues are apparently disordered in all custal structures and remain unresolved, which is consistent with the presence of four GGM repeats in the equatorial domain's sequence. Electron microscopy reconstructions [27] and small-angle neutron scattering studies [28] showed additional density in the central channel, on the level of the equatorial domains, which might account for these residues. The nucleotide-binding site of each subunit is located at the top of the equatorial domain, facing the central channel. It contains a set of conserved loops including the G D G T T sequence, a motif that is highly conserved among all chaperonins. The crystal structure of GroEL with bound ATPyS revealed that this sequence is a phosphate-binding loop that coordinates with all the phosphate oxygens either directly or indirectly via magnesium ions [29].
Apical domain The apical domain forms the other end of the G r o E b cylinder. It contains the binding sites both for substrate protein and co-chaperonin and is the least traceable part of the GroEL structure. The high atomic temperature factors in this area, combined with the observed differences between noncrystallographic symmetry-related molecules, suggest an intrinsic flexibility [30] that appears to be necessary for accommodating a wide range of substrate proteins on its hydrophobic surface. The st, bstrate proteins span a wide spectrum of molecular mass and chemico-physical properties [31,32"], defying a rigid lock-key model for interaction. T h e necessary degree of structural flexibility can be reached either globally, through rigid-body movements of the entire domain (for different sobstrate sizes), or locally on the channel surface, through mobile loops (for different protein conformations). It is assumed that the general substrate recognition and binding mechanism is based on hydrophobic interactions [33"].
Intermediate domain T h c smallest of the three domains, called the intermediate domain, seems to act mainly as a linker between the equatorial and the apical domains. Hinge-like connections to both domains on either side of the intermediate domain allow for the large rigid-body movements observed in electron microscopy reconstructions [34] and in a comparison of the crystal structure of the GroEL-GroES complex [35"] with the structure of GroEL alone. The main purpose of this domain seems to be the transmission of allosteric changes between the equatorial and apical domains. This hypothesis is further supported by the fact that salt bridges formed between the intermediate domain and the apical domain of adjacent subunits play a crucial role in the observed positive cooperativity of nucleotide binding and hydrolysis within one ring, and in the negative cooperativity between rings [36,37"',38]. Also, Fenton eta/. [39] observed that mutations in the intermediate domain affect all of the measurable functions of GroEL, which supports the view that the intermediate domain plays a major role in signal transmission.
GroES The co-chaperonin GroES forms a single ring structure and is composed of seven subunits each with a molecular weight of 10 kDa. The crystal structure of GroES alone shows a hollow dome-shaped structure with an outer diameter of 70-80 A, a height of 30 A, and an inner core 30]X in diameter and 20 A in height [40,41]. The central structural element in each subunit is a [3 barrel with two [8-hairpin loops: one reaching upwards to form the top of the dome, the other protruding from the base in order to interact with its chaperonin partner, GroEL. The loops involved in GroEL binding are flexible and could not be resolved in the crystal structure [40]. A comparison of the structure of the asymmetrical G r o E L - G r o E S complex with that of GroEL alone reveals two large rigid-body movements of entire domains [35"°]. Upon binding GroES, the apical domain of GroEL moves upwards out of the equatorial plane by 60 °, while twisting around by 90 °. This opening of the apical domains almost doubles the volume of the cavity inside GroEI,, which also becomes continuous with the dome of GroES. At the same time, the intermediate domain shifts by 25' towards the equatorial domain (Figure 3). T h e latter movement brings residue D398 of the intermediate domain as an additional ligand into hydrogen-bonding distance with the magnesium ion at the nuclentide-binding site. Mutational studies suggest that D398 is involved in ATP hydrolysis [42].
Reaction pathway of G r o E L - G r o E S m e d i a t e d folding In spite of the considerable research effort directed towards explaining the mechanism by which GruEL assists folding, little is known about the fate of a polypeptide undergoing a cycle of chaperonin-mediated protein folding. A basic model seems to be in place but interpretations sometimes vary to the point of giving rise to fierce debate. Based on the observation that polypeptides appear to be held within the central cavit'~; it has been suggested that GroEL performs its flmction by providing a so-called 'box of infinite dilution' [43] in which the substrate protein folds while awfiding unproductive interactions with other unfolded proteins. According to this model, release of the polypeptide would occur after it has reached its native state. In successflfl refolding experiments with isolated apical domains (residues 191-345), Zahn et a/. [22"] demonstrated that neither the presence of a central cavity nor the allosteric properties of the GroEL complex are essential for successful binding and refolding in vitro. They assume that the allosteric changes observed upon binding of GroES and ATP regulate the affinity of the complex for substrates in vivo. Jackson eta/. [44] proposed a model in which GroEL binds and unfolds aggregation-prone or kinetically trapped fold-
Chaperonins Braig
Figure 3
(a)
4o
4o
(b) GroES
i#
2°
2o
CurrentOpinionin StructuralBiology Schematic representation of the rigid-body movements in the GroEL complex upon GroES binding. (a) Before GroES binding, the interface between the two rings of the GroEL complex is slightly deformed. The subunits of the cis ring are tilted inwards by about 4 °, while the subunits of the trans ring tilt outwards by approximately 2 °. The complementary movement of the trans ring is necessary in order to preserve the inter-ring contacts in the equatorial plane [35"]. (b) Binding of GroES is accompanied by two major hinge movements (H t and H2). The intermediate (I) domains move down towards the equatorial (E) plane by approximately 25 ° thus closing the nucleotidebinding pockets. The apical (A) domains of the cis ring swing upwards by some 60 ° relative to the equatorial plane. At the same time they twist around their long axis by 90 ° [34]. This movement brings the apical domains in close proximity to the mobile loops on the underside of GroES that form the interface between GroEL and GroES [35"].
ing intermediates. Studies using a release-deficient mutant of GroEL [45] suggest that ATP binding promotes the release of non-native proteins into the bulk solution, where they either fold to the native state, or rebind as a kinetically trapped intermediate to another GroEL molecule and assume their original unfolded state [7°',46,47]. Recent results using either undiluted Xenopus egg lysates or macromolecular crowding agents to mimic the physicochemical properties of the prokaryotic cytosol contradict the release-rebind model [48"'].
Conclusions Even though the protein folding pathways of the two major chaperone systems involved in protein folding are globallx understood, there is much debate about single aspects of the process. Simple outlines seem to be in place and some of the complexities of the process of chaperonin-mediated protein folding have yielded to structural and biochemical investigations while others remain obscure. This is particularly true when it comes to identifying the nature of polypeptide binding and concomitant fi)lding, and explaining the active participation of the cavity in the cis position relative to GroES in directing productive folding within it.
163
Abundant data produced by the many groups working in the field seem to support different models. Upon closer inspection, it turns out that the interpretations depend to a large extent on the definitions and experimental procedures used to mimic the conditions in the living cell. The further we go in our understanding of chaperone-mediated protein folding the more careful we have to be in minute details and the more important it is to reach an eventual consensus. One of the main problems in applying a single model to describe the function of chaperonins for all substrate proteins is the great variety of potential substrates: they vary greatly in the tendency of their compact intermediates to aggregate, and also in their rate of spontaneous refolding. One should always keep in mind that most in vitro studies have been carried out Using only a few selected substrate proteins, namely dehydrofolate reductase, malate dehydrogenase and rhodanase. The generality of conclusions based on these experiments remains to be firmly established.
Acknowledgements T h e author thanks Eric de la Fortelle and C h a p i n A Rodriguez for their critical reading and discussion of the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ° of outstanding interest 1.
Anfinsen CB: Principles that govern the folding of protein chains. Science 1973, 181:223-257.
2.
Buchner J: Supervising the fold: functional principles of molecular chaperones. FASEB J 1996, 10:10-19.
3.
Hartl FU: Molecular chaperones in cellular protein folding. Nature 1996, 381:571-580. An excellent introduction to the field of molecular chaperones and assisted protein folding, with special emphasis on the hspT0 family and GroEL. 4.
Ellis RJ: Proteins as molecular chaperones. Nature 1987, 328:378379.
5.
Frydman J, Hohfeld J: Chaperones get in touch: the Hip-Hop connection. Trends Biochem Sci 1997, 22:87-92.
6.
Buchberger A, Schroder H, Hesterkamp T, Schonfeld HJ, Bukau B:
•.
Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding. J Mo! Biol 1996, 261:328-333.
Studies with denatured luciferase show that DnaK and GroEL compete for binding to non-native proteins; therefore, in this folding reaction, DnaK and GroEL do not necessarily act in succession by promoting earlier or later folding steps, but rather form a lateral network of proteins. 7.
Farr GW, Scharl EC, Schumacher RJ, Sondek S, Horwich AL:
•.
Chaperonin-mediated folding in the eukaryotic cytosol proceeds through rounds of release of native and nonnative forms. Cell
1997, 89:927-937. This study examines the fate of newly synthesized cytosolic proteins bound to CCT in reticulocyte lysate and Xenopus oocytes. In both cases, the production of the native protein is strongly inhibited by the introduction of a chaperonin trap, which is able to bind but not to release substrate protein. The overall mechanism of CCT-assisted protein folding resembles that of GroEL (see annotation [48"']). 8.
Lewis VA, Heynes GM, Zheng D, Sailbil H, Willison K: T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol. Nature 1992,358:249-252.
164
9.
Macromolecular assemblages
Phipps BM, Typke D, Hegerl R, Volker S, Hoffmann A, Stetter KO, Baumeister W: Structure of a molecular chaperone from a thermophUic archaebacterium. Nature 1993, 361:475-477.
26. Braig K, Otwinowski Z, Hedge R, Boisvert DC, Joachimiak A, Horwich AL, Sigler PB:.The crystal structure of the bacterial chaperonin GroEL at 2.8 A resolution. Nature 1994, 371:578-586.
10. TrentJD, Nimmesgern E, Wall J, Hartl FU, Horwich AL: A molecular chaperone from the thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature 1991, 354:490-493.
27. Chen S, Roseman AM, Hunter AS, Wood SP, Burston SG, Ranson NA, Clarke AR, Saibil HR: Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryoelectron microscopy. Nature 1994, 371:261-264.
11. Kubota H, Hynes G, Willison K: The chaperonin containing tcomplex polypeptide 1 (TCP-1) multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. Eur J Biochem 1995, 230:3-16.
28. Thiyagarajan P, Henderson SJ, Joachimiak A: Solution structures of GroEL and its complex with rhodanase from small-angle neutron scattering. Structure 1996, 4:?9-88.
12. Knapp S, Schmidt-Krey I, Herbert H, Bergman T, Jornvall H, Ladenstein R: The molecular chaperonin TF55 from the thermophiiic archeon Sulfolobus solfataricus. A biochemical and structural characterization. J Me~ Bio/1994, 242:397-407. 13. Rommelaere H, van Treys M, Gao Y, Melki R, Cowan NJ, Vandekerckhove J, Ampe C: The eukaryotic cytosolic chaperonin contains t-complex polypeptide and several related subunits. Proc Nat/Acad Sci USA 1993, 90:11975-11979. 14. Kubota H, Hynes G, Came A, Ashworth A, Willison K: Identification of six Tep-1 related genes encoding divergent subunits of the TCP-l-containing chaperonin. Curr Bio/1994, 4:89-99. 15. Gao Y, Thomas JO, Chow RL, Lee GH, Cowan NJ: A cytoplasmic chaperonin that catalyzes ~-actin folding. Cell 1992, 69:10431050. 16. Lewis SA, Tian G, Vainberg IE, Cowan NJ: Chaperonin-mediated folding of actin and tubulin. J Cell Biol 1996, 132:1-4. 17. YaffeMB, Farr GW, Miklos D, Horwich AL, Sternlicht ML, Sternlicht H: TCP1 complex is a molecular chaperone in tubulin biogenesis. Nature 1992, 358:245-258. 18. Frydman J, Nimmesgern E, Ohtsuka K, Hartl FU: Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 1994, 353:111-117. 19. Sternlicht H, Farr GW, Sternlicht ML, Driscoll JK, Willison K, Yaffe M: The t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo. Proc Natl Acad Sci USA 1993, 90:9422-9426. 20. Roobol A, Holmes FE, Hayes NVL, Baines A J, Carden AJ: Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells. J Cell Sci 1995, 108:1477-1488. 21. Braig K, Simon M, Furuya F, Hainfeld JF, Horwich AL: A polypeptide bound by the chaperonin groEL is localized within a central cavity. Proc Nat/Acad Sci 1993, 90:3978-3982. 22. Zahn R, Buckle AM, Perret S, Johnson CM, Corrales FJ, Golbik R, Fersht AR: Chaperonin activity and structure of monomeric polyeptide binding domains of GroEL. Proc Nat/Acad Sci USA 1996, 93:15024-15029. The crystal structure of a monomeric polypeptide that corresponds to the apical domain of GroEL shows a well-ordered structure with the same fold as that of native GroEL. The isolated domain functions as a minichaperone (see annotation [33"]). 23. GroB M, Robinson CV, Mayhew M, Hartl FU, Radford SE: Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling. Protein Sci 1996, 5:2506-2513. Structural analysis using hydrogen exchange labeling reveals that during several cycles of GroEL-assisted folding of dihydrofolate reductase the protein is partially folded rather than being completely unfolded upon binding to GroEL. 24. Goldberg MS, Zhang J, Sondek S, Matthews CR, Fox Re, Horwich °. AL: Native-like structure of a protein-folding intermediate bound by the chaperonin GroEL. Proc Natl Acad Sci USA 1997 94:10801085. The structure of DHFR bound to GroEL was analyzed using hydrogen-deuterium exchange and NMR spectroscopy. The data indicate that central structural elements found in the native protein are also present in the GroEL-bound form of the protein. Since these structural elements are derived from distant parts of the primary structure, the authors conclude that a native-like global topology is present in folding intermediates that are bound to GroEL. 25. Lille H, Buchner J: Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding. Proc Natl Acad Sci USA 1995, 92:8100-8104.
29. Boisvert DC, Wang J, Otwinowski Z, Horwich AL, Sigler PB: The 2.4/~, crystal structure of the bacterial chaperonin GroEL complex with ATPIsS.Nat Struct Biol 1996, 3:1 ?0-17?. 30. Braig K, Adams P, Brunger AT: Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution. Nat Struct Biol 1995, 2:1083-1093. 31. Ewalt KL, Hendrick JP, Houry WA, Hartl FU: In rive observation of polypeptide flux through the bacterial chaperonin system. Ceil 199?, 90:491-500. 32. LorimerGH: A quantitative assessment of the role of chaperonin proteins in protein folding in vivo. FASEB J 1996, 10:5-9. "What proportion of all the proteins of Escherichia coil reach their native states with the assistance of the chaperonins proteins, GroEL and GroES?" The author provides an easy to follow calculation to answer that question for a given E. coil strain under standard conditions. 33. Buckle AM, Zahn R, Fersht AR: A structural model for GroELpolypeptide recognition. Proc Nail Acad Sci USA 1997, 94:35713575. This paper presents the high resolution structure of an N-terminal-tagged polypeptide corresponding to the isolated apical domain of GroEL. In the structure, the N-terminal tag of one molecule is bound to the active region of a neighbouring molecule. The data presented here are used to reconstruct how a peptide can bind to the GroEL complex. 34. RosemanAM, Chen S, White H, Braig K, Saibil HR: The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell 1996, 87:241-251. 35. Xu Z, Horwich AL, Sigler PB: The crystal structure of the •. asymmetric GroEL-GroES-(ADP) 7 chaperonin compex. Nature 199'7, 388:741-750. This landmark paper presents the long awaited crystal structure of the asymmetrical (GroES)7-(GroEL)14 complex. The structure shows that there is a massive upward movement of the GroEL apical domains, which is accompanied by a twisting rigid-body movement around a hinge at the junction of the intermediate and apical domains. It further reveals that as a result of these movements, the surface properties of the central cavity in the cis cavity dramatically change from hydrophobic to polar. If one assumes that protein binding is due to hydrophobic interactions, GroEL would lose its binding properties once the cavity becomes polar. 36. Yifrach O, Horovitz A: Two lines af allosteric communication in the oligomeric chaperonin GroEL are revealed by the single mutation Arg196/Ala. J Mol Biol 1994, 234:397-401. 37. White HE, Chen S, Roseman AM, Yifrach O, Horovitz A, Saibil HR: •Structural basis of the allosteric changes in the GroEL mutant Arg 197 --) Ala. Nat Struct Biol 1997, 4:690-694. Cryo-electron microscopy has been used to generate three-dimensional reconstructions of a GroEL mutant with weaker intersubunit contacts, in different nucleotide-bound states. In this mutant, the domains are more free to move about the intermediate domain at all ATP concentrations. The study provides new insights into the nucleotide-dependent allosteric switching of GroEL. 38. Aharoni A, Horovitz A: Detection of changes in pairwise interactions during allosteric transitions: Coupling between local and global conformational changes in GroEL Proc Natl Acad Sci USA 199"7, 94:1698-1702. 39. Fenton WA, Kashi Y, Furtak K, Horwich AL: Residues in chapronin GroEL required for polypeptide binding and release. Nature 1994, 371:614-619. 40. Hunt JF, Weaver AJ, Landry SJ, Gierasch L, Deisenhofer J: The crystal structure of the GroES co-chaperonin a~ 2.8 A resolution. Nature 1996, 379:37-45.
Chaperonins Braig
165
41. Manda SC, Mehra V, Bloom BR, Hol WGJ: Structure of the heat shock protein chaperonin-10 of Mycobacterium leprae. Science 1996, 271:203-207.
49. Nelson RJ, Ziegelhoffer T, Nicolet C, Werner-Washburne M, Craig EA: The translation machinery and 70 kDa heat shock protein cooperate in protein synthesis. Ceil 1992, 71:97-105.
42. Rye HS, Burston SG, Fenton WA, Beechem JM, Xu Z, Sigler P, Horwich AL: Distinct action of cis and trans ATP within the double ring of the chaperonin GroEL Nature 1997, 388:792-798.
60. Reid BG, Flynn GC: GroEL binds to and unfolds rhodanase pos~ranslationally. J Biol Chem 1996, 271:7212-7217.
43. Ellis RJ: Opening and closing the Anfinsen cage. Curr Biol 1994, 4:633-635.
51. Hesterkamp T, Hauser S, Lutcke H, Bukau B: Escherichia colitrigger factor is a propyl isomerase that associates with nascent polypeptide chains. Proc Nat/Acad Sci USA 1996, 93:4437-4441.
44. JacksonGS, Staniforth RA, Halsall DJ, Atkinson T, Holbrook JJ, Clarke AR, Burston SG: Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: Implications for the mechanism of assisted protein folding. Biochemistry 1993, 32:2554-2563.
52. ValentQA, Kendall DA, High S, Kusters R, Oudega B, Luirink J: Early events in preprotein recognition in E. coil: interaction of SRP and trigger factor with nascent polypeptides. EMBO J 1995, 14:54945505.
45. Weissman JS, Kashi Y, Fenton WA, Horwich AL: GroEL mediated protein folding proceeds by multiple rounds of binding and release of non-native forms. Ceil 1994, 78:693-702. 46. Ranson NA, Dunster NJ, Burston SG, Clarke AR: Chaperonins can catalyse the reversal of early aggregation steps when a protein misfolds. J Mo/ Bio/1995, 250:581-586. 47
Burston SG, Weissman JS, Farr GW, Fenton WA, Horwich AL: Release of both native and non-native proteins from cis-only GroEL ternary complex. Nature 1996, 383:96-99.
48. Martin J, Hartl FU: The effect of macromolecular crowding in o. chaperonin-mediated protein folding. Proc Nat/Acad Sci USA 1997, 94:1107-1112. The point of this study was to investigate the fate of newly synthesized proteins in a dense cytosolic solution or in the presence of macromolecular crowding agents. Under these conditions, the transfer to a chaperonin trap, which is able to bind but not to release substrate protein, is prevented. At a twofold dilution, a significant increase in the rate of transfer is observed. The authors come to the conclusion that the release of non-native polypeptide is not an essential feature of the productive chaperonin mechanism. See annotation [7"'].
53. Langer T, Lu C, Echols H, Flanagan J, Hayer MK, Hartl FU: Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 1992, 356:683-689. 54. Gragerov A, Nudler E, Komissarova N, Gaitanaris GA, Gottesman ME, Nikiforov V: Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia co//. Proc Nat/Acad Sci USA 1992, 89:10341-10344. 55. Bukau B, Walker GC: Cellular defects caused by deletion of the Escherichia coil DnaK gene indicate roles for heat shock protein in normal metabolism. J Bacterio/1989, 171:2337-2346. 56. Spatter H, Buchner J: How GroES regulates binding of nonnative protein to GroEL. J Bio/Chern 1997, 272:14080-14086. This kinetic study suggests that the slow release of substrate protein from the unproductive trans position in the asymmetric GroEL-GroES complex favours the binding of a second GroES. The formation of a symmetric complex ensures that the substrate protein is sequestered in a position underneath GroES. According to this model, the intrinsic binding characteristics of the asymmetric trans complex determine the sequence of events during the reaction cycle.