JORG MARTIN AND F ULRICH HARTL MINIREVIEW

Protein folding in the cell: molecular chaperones pave the way

Structure 15 November 1993, 1:161-164

In vitro, many unfolded polypeptides are able to fold to the native state spontaneously, indicating that the amino acid sequence of a protein contains all the information necessary to specify its three dimensional conformation [ 1 ]. It had been assumed that protein folding in vivo also generally occurs in a spontaneous process. This view has changed only recently due to the discovery of a number of proteins, now commonly called 'molecular chaperones', which are essential for cellular protein folding and occur ubiquitously in eubacteria, archaebacteria and in eukary otic cells [2].

Different classes of molecular chaperones share the prop erty of interacting with other proteins in their non-native conformations, apparently by recognizing the hydropho bic sequences or surfaces which are exposed to solvent in the unfolded, but not in the native, state [3-5]. Molecular chaperones do not interact with a specific consensus se- quence motif and thus have the ability to prevent incorrect intra- and inter molecular folding and association of many different proteins. The physiological significance of this function can easily be understood given the high concen- tration of folding polypeptide chains in the cell, which would strongly favour protein aggregation. It should be stressed that molecular chaperones increase the yield of correctly folded protein by suppressing off pathway reac tions that compete with the productive folding pathway, but do not act as folding catalysts. The major rate-limiting transition state of protein folding remains unchanged in the presence of chaperones. To fulfil their task, molec ular chaperones bind to the nascent polypeptide as it emerges from the ribosome and accompany it along its folding pathway (Fig. 1). Their duties do not end once the native state has been reached, however. These versatile helper proteins are able to interact again with their former proteges should they expose appropriate binding sites due to unfolding [2]. The possibility of protein denatu- ration and aggregation is increased under cellular stress conditions, such as elevated temperature. Indeed, many molecular chaperones have been classified as stress or heat shock proteins (Hsps) whose expression is induced under cellular stress.

The functions of the chaperone proteins of the Hsp70 and Hsp60 families are most intimately related with pro- tein folding under both normal and stress conditions. Al- though substrate binding and release by both of these classes of proteins is dependent on ATP binding and hydrolysis, two distinct principles of chaperone action can be distinguished. The Hsp70s act as monomers of about 70 kDa, or as dimers. Their predominant role appears to

be to shield the hydrophobic surfaces of nascent polypep tides until at least a complete protein domain (100-200 amino acid residues in length) is available for produc tive folding. The Hsp60s on the other hand form large double-ring complexes with an internal cavity which can sequester a completely synthesized, bur as yet unfolded protein (or protein domain) Dora the cellular milieu. They prevent protein aggregation and at the same time allow folding to the native state to proceed in an ATP-dependent reaction. HspT0s and Hsp60s can function in a sequential pathway of chaperoned folding [61 which is depicted in Fig. 1.

The mechanism of action of the Hsp70s and Hsp60s has been analyzed by both genetic and biochemical ap- proaches, in particular by reconstituting the folding pro cess in vi trowith purified components [6-8]. Two sets of cytosolic chaperone proteins from Escherichia coli were employed in these studies: (a) DnaK (tIsp70) together with its partner chaperone DnaJ and the regulator protein GrpE, and (b) GroEL (Hsp60) with its regulator GroES. DnaK, DnaJ and their eukaryotic counterparts are able to interact with nascent polypeptides emerging from ribo- somes. They have been co-purified with polyribosomes and have been shown to bind co translationally to grow- ing polypeptide chains (A Gragerov, personal commu- nication) [9-11]. DnaJ recognizes ribosome-bound se- quences as short as 55 amino acid residues [10]. Asso- ciation of the 41 kDa chaperone protein DnaJ with the unfolded polypeptide plays a role in targeting the sub strate protein to DnaK, which leads to the formation of a stable ternary complex. In this 'm~nage ~ trois' DnaJ stabilizes the M)P-bound state of DnaK which has high binding affinity for unfolded protein. Analysis of the bind- ing specificity of Hsp70 has demonstrated a preference for short, extended peptide segments within a polypeptide chain which are enriched in hydrophobic amino acids [3,5]. Optimal binding is obtained with a minimum pep- tide length of seven residues [3]. The peptide binding pocket of Hsp70 is most probably located within about 25 kDa of the carboxyl terminus of the protein; it has been proposed that this pocket has a structure similar to the binding cleft of the MHC class I histocompatibility glycoprotein [21. The amino terminal domain (of about 40 kDa) of Hsp70 has an actin like fold [2] and contains the ATP-binding site. The substrate specificity of DnaJ has not yet been analyzed in detail but it appears to be distinct from that of DnaK. In addition to preventing misfolding of the growing polypeptide chain, binding of Hsp70 is apparently important for efficient translation itself. In the absence of functional Hsp70, newly synthesized polypep-

Current Biology Ltd ISSN 0969-2126 161

162 Structure 1993, Vo] "1 No 3

(a) DnaK and DnaJ interact with a nascent polypeptide chain emerging from the ribosome.

(b) A ternary complex containing DnaK, Dnat and unfolded protein is formed. Dnal stimulates the ATPase activity of DnaK and stabilizes DnaK in the ADP-state.

N

Pi

ADP

(c) GrpE induces the release of ADP followed by the binding of ATP. The partially-folded polypeptide is released and transferred to a GroEL/GroES complex with GroEL having bound ADP tightly.

Fully-folded protein

(f) Once folded, the substrate protein has lost its affinity for the chaperonin and leaves the GroEL cavity.

ADP +Pi

ATP

ATP

(e) During multiple rounds of substrate protein release and rebinding, GroES can cycle between the two ends of the GroEL cylinder.

ADP +Pi

(d) Binding of the folding intermediate in the central cavity of GroEL leads to dissociation of ADP and GroES. As a consequence of ATP rebinding and subsequent ATP hydrolysis, the substrate protein is released into the GroEL cavity for folding.

Fig. 1. Model for the pathway of chaperone mediated protein folding

tide chains may be unable to ex i t eff iciently f rom the ri bosome, thus jamming the ribosomal exit tunnel [11].

While binding of exposed hydrophobic polypeptide seg- ments to DnaK/DnaJ can prevent protein aggregation, folding to the native state requires that the bound strut tures be released. Release is the consequence of confor- mational changes in DnaK which occur upon ATP binding and hydrolysis. This is dependent on the regulation of DnaK by the 20 kDa Hsp GrpE (A Szabo and FU Hartl, unpublished data) [6] which functions as a nucleotide exchange factor. DnaK has a low ATPase activity which is regulated by DnaJ so that the complex of DnaK, DnaJ and unfolded protein contains tightly bound ADP. GrpE interacts with the ATPase domain of DnaK and induces the release of ADP. This is followed by ATP binding to DnaK, whereupon the ternary complex dissociates. No- tably, it is the binding of ATP and not its hydrolysis, which

in the cytoso[ of Escherichia coll.

leads to protein release (A Szabo and FU Hartl, unpub lished data; DR Palleros, KL Reid, L Shi, WJ Welch and FL Fink, personal communication). The released polypeptide may then be able to fold to the native state. In many cases, however, efficient folding may not be achieved by the Hsp70 chaperone system alone. Rather, the polypep- tide substrate is maintained in a non-native conformation which cycles between chaperone-bound and free states. These multiple rounds of protein release and rebinding require ATP hydrolysis by Hsp70.

Upon release from DnaK/DnaJ, the folding polypeptide may be transferred to GroEL (Hsp60), which provides an isolated compartment in which folding can proceed. The unusual quaternary structure of the GroEL complex may explain this ability: 14 subunits each of 60 kDa which are arrranged in two stacked rings with seven-fold wm- metry, forming a symmetrical double toroid with a large

Molecular chaperones Martin and Hartl 163

central cavity [12,13]. Electron microscopy indicates that one or two molecules of substrate protein bind within this space, which can accommodate polypeptides up to 90kDa [12,14]. When associated with GroEL in the ab- sence of ATP, the substrate protein is stabilized in the compact 'molten globule' state [7]. Functional analysis of GroEL supports the idea that at least the partial forma tion of ordered tertiary structure occurs within the GroEL cylinder, thus effectively preventing unproductive interac- tions within and between folding polypeptides (J Martin and FU Hartl, unpublished data) [7]. Under physiologi cal conditions, GroEL probably exists as a complex with GroES, a heptameric ring of identical 10 kDa subunits. A single GroES heptamer binds to one end of the GroEL cylinder conferring asymmetry to the chaperone complex [12]. As in the case of Hsp70, polypeptide binding and release by GroEL is regulated by an ATPase activity [2,7]. Formation of a complex between GroEL and GroES is dependent on nucleotide binding and leads to the sta bilization of GroEL in the ADP state. The folding pro- tein may enter the cavity of GroEL at the end of the double toroid that is not bound to GroES. It is believed that the association with the chaperone involves multi pie attachment sites within the polypeptide chain which make contact with binding regions of the individual GroEL subunits. NMR studies with short peptides have indicated that GroEL may recognize elements of secondary structure in partially-folded proteins [4]. High affinity binding to GroEL may require the presence of a polypeptide segment that is large enough to allow contact with more than one GroEL subunit. This would be consistent with the obser- vation that GroEL does not interact efficiendy with nascent polypeptide chains and is not co-isolated with translating polyribosomes (A Gragerov, personal communication).

Protein binding to GroEL in the absence of ATP prevents folding, except perhaps for certain small proteins such as barnase (11 kDa) which interact weakly and may fold while maintaining a loose association with the chaperone [15]. This raises the question of how GroEL normally allows protein release but at the same time ensures re- tention of the polypeptide until folding has progressed sufficiently to circumvent the fatal danger of aggregation. Recent experiments indicate that the substrate protein triggers its own release by inducing exchange of ADP for ATP (J Martin and FU Hard, unpublished data). Upon pro- tein binding, the "affinity of GroEL for ADP is decreased. As a consequence, GroES dissociates, making the seven subunits in the interacting GroEL toroid accessible for ATP binding. This in turn reduces the affinity of GroEL for substrate protein and may allow the partial or corn plete release of polypeptides which bind with low affinity. Tightly-bound proteins such as mitochondrial rhodanese depend on ATP hydrolysis for their release from GroEL [2,7]. GroES reassociates with GroEL in the ATP state prior to ATP hydrolysis. The time between ATP binding to GroEL and the regeneration of the ADP state would be the period available to the substrate protein for fold- ing. If upon a single release event the protein has not yet reached the native state, a folding intermediate still exposing appropriate recognition elements would rebind to GroEL, thus inducing another cycle of release and fold-

ing. Multiple rounds of interaction with GroEL may be required for most proteins to fold and finally leave GroEL. Rhodanese folding, for example, requires the hydrolysis of about 130 molecules of ATP by GroEL [7]. GroES has a pivotal function in this process. Regulated by GroES, the GroEL subunits in an individual ring will hydrolyze ATP cooperatively (J Martin and FU Hard, unpublished data) [7]. This would seem necessary to allow the more or less simultaneous release of a substrate protein from its multiple attachment sites for folding. Only in the pres ence of GroES is the folding substrate protein shielded from the bulk solution and segregated from other dena- tured proteins [7]. Although it seems clear that the co- ordinating effect of GroES is responsible for this protec tion, the structural basis of this remarkable effect remains to be determined. An attractive possibility is that GroEL adopts a conformation where access to its central cavity is sterically hindered during ATP hydrolysis, when the sub strate protein is released for folding. In a simplistic view the main function of GroEL could, thus, be to provide a microenvironment in which intermolecular associations are prevented and cellular protein folding can occur as under conditions of 'infinite dilution'. (Under these con- ditions, a single protein molecule would be allowed to pursue its folding potential without being in danger of interacting unproductively with another folding polypep tide.)

It remains to be seen whether the two distinct functional principles of the Hsp70 and Hsp60 chaperone systems are of general importance for protein folding in different cell types and compartments. Evidence that they are is provided by the recent finding that the eukaryotic cytosol not only contains Hsp70, but also DnaJ homologues and the TCP-1 ring complex, which shares structural and func tional features with the Hsp60s [2]. Further insight into the mechanisms by which molecular chaperones mediate protein folding will depend on the progress of several crystallographic laboratories in obtaining the necessary structural information on these fascinating components.

Acknowledgement'. The authors thank Roman Hlodan Ebr critically reading the manuscript

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J 6 r g M a r t i n a n d F U l r i c h Hart l , Ce l l u l a r B i o c h e m i s t r y a n d B i o p h y s i c s P r o g r a m , M e m o r i a l S l o a n - K e t t e r i n g Can - c e r C e n t e r , 1275 Y o r k A v e n u e , N e w Y o r k , NY 10021 , USA.