p272

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F Ulrich Hartl & Manajit Hayer-Hartl Converging concepts of protein folding in vitro and in vivo

Nsb  16 , 574 (2009)

* Folding intermediates are the rule for larger proteins of $\sim$100 amino acids (>90% of all proteins in a cell), which have a greater tendency to rapidly collapse in aqueous solution into compact non-native conformations (cf. Brockwell, D.J. & Radford, S.E. Intermediates: ubiquitous species on folding energy landscapes? Curr. Opin. Struct. Biol. 17, 30 (2007).)

*Thermodynamically highly stable structures (e.g., aggregates and amyloid structures) are accessible to many proteins under denaturing conditions, largely independent of sequence, suggesting that their formation is an inherent property of the polypeptide chain.

*Folding occurs in the presence of 300-400 g/l of protein and other macromolecules.

*Notably, a number of essential proteins have extremely low intrinsic folding efficiencies and essentially do not fold in the absence of chaperones. For example, actins and tubulins seem to have highly energetically frustrated folding pathways and can overcome kinetic folding barriers only through assistance by chaperones.

* Numerous classes of structurally unrelated chaperones have been described. The cellular chaperone machinery forms complex networks that are indispensable for protein quality control and maintenance of protein homeostasis.

*The chaperone pathways and networks acting in protein folding in the cytosol follow general organizational rules. In all three domains of life, there are two major principles of chaperone action:

(i) machinery that functions in stabilizing nascent polypeptides on ribosomes and initiating folding and

(ii) components that act downstream in completing the folding process (Fig. 3). 

The number of interacting substrates and the degree of functional redundancy among chaperone components decreases from upstream to downstream.

The first category of factors includes chaperones that bind directly to the large ribosomal subunit in close proximity to the polypeptide exit site, such as bacterial Trigger factor (Fig. 3a) and a specialized Hsp70 system called RAC (ribosome-associated complex) in eukaryotes (Fig. 3c). Additionally, archaea and eukarya contain the nascent chain-associated complex, NAC. They bind linear chain segments enriched with hydrophobic amino acids.

*Members of the Hsp70 family (DnaK in bacteria, Hsc70 in higher eukaryotes Fig. 5) function as second-tier chaperones for longer nascent chains. These factors do not bind directly to the ribosome and mediate co- or post-translational folding through ATP-regulated binding cycles. 

*Co-translational domain folding serves to avoid non-native interdomain contacts, thus smoothing the energy landscape for large proteins. This mechanism is essential for the folding of the large number of eukaryotic multidomain proteins and is facilitated by the slower translation speed in eukaryotes ($\sim$4 amino acids per second versus $\sim$20 amino acids per second in bacteria).

* Trigger factor and DnaK have partially overlapping functions, but their combined deletion is lethal at temperatures above 30$^\circ$ for  E. coli . (Fig. 4)

*Trigger factor is an abundant $\sim$50-kDa protein (Fig. 4a). Trigger factor exists in two forms: a monomer when bound to the ribosome and a dimer when free in the cytosol. The N domain binds to ribosomal proteins L23 and L29 next to the polypeptide exit site54, with a mean residence time of 10-15s (ref. 19). When the nascent chain exposes strongly hydrophobic segments, Trigger factor leaves the ribosome but remains associated with the elongating chain.

*The chaperone pathways operating in ER follow analogous organizational principles, but specialized machinery is used in disulfide bond formation and sugar modification of many secretory proteins.

*The biogenesis and folding of membrane proteins uses specialized machinery for insertion and assembly of the membrane-integrated parts, whereas cytosolic and ER luminal chaperones assist in the folding of exposed domains (This same issue contains a review on this).

The Hsp70 system  (Fig. 5). The Hsp70 proteins are the most versatile chaperones and occur both as constitutively expressed and stress inducible forms25. Besides broadly assisting in de novo folding, they have various other functions, including protein trafficking and assistance in the proteolytic degradation of terminally misfolded proteins.

*Hsp70s generally collaborate with chaperones of the Hsp40 (DnaJ) family and nucleotide exchange factors (NEFs) in the ATP-regulated binding and release of non-native proteins60. Their role in de novo folding begins by binding to nascent chains, but they generally do not interact directly with the ribosome. Binding and release by Hsp70 is achieved through the allosteric coupling of a conserved N-terminal ATPase domain ($\sim$40 kDa) with a C-terminal peptide-binding domain (PBD) ($\sim$25 kDa), the latter consisting of a $\b$-sandwich subdomain and an $\a$-helical lid segment61 (Fig. 5a). The $\b$-sandwich recognizes extended, $\sim$7-residue segments enriched with hydrophobic amino acids62. Such segments occur on average every 50–100 residues in proteins. The $\a$-helical lid and a conformational change in the $\b$-sandwich domain regulate the affinity state for peptide in an ATP-dependent manner60,63. In the ATP-bound state, the lid adopts an open conformation, resulting in high on- and off-rates (low affinity) for peptide. Hydrolysis of ATP to ADP is strongly accelerated by Hsp40, leading to lid closure and stable peptide binding (low on- and off-rates; high affinity) (Fig. 5b).

*Binding of Hsp70 to non-native substrate hinders aggregation by transiently shielding exposed hydrophobic segments and at the same time reducing the concentration of aggregation-prone species. 

The chaperonins  (Fig. 6) Chaperonins are large, double-ringed complexes of $\sim$800 kDa. There are two groups of chaperonin26,71. Members of group I (also called Hsp60s) occur in bacteria (GroEL), mitochondria and chloroplasts. They have seven-membered rings and functionally cooperate with Hsp10 proteins (bacterial GroES), which form the lid of the folding cage. The group II chaperonins in archaea (thermosome) and in the eukaryotic cytosol (TRiC/CCT) consist of eight- or nine-membered rings. They are independent of Hsp10 factors, their lid function being built into the chaperonin ring in the form of specialized $\a$-helical extensions.

*GroEL-bound substrates populate an ensemble of compact and locally expanded states that lack stable tertiary interactions (Fig. 6b). Binding of GroES is preceded by ATP binding to GroEL and causes a pronounced conformational change that leads to the formation of a cage with a highly hydrophilic, net negatively charged inner wall. Encapsulated protein up to $\sim$60 kDa is free to fold in this environment for 10–15 s, the time needed for ATP hydrolysis in the GroES-bound ring (cis-ring). Protein substrate leaves the cage upon GroES dissociation, which is induced by ATP binding in the opposite ring (trans-ring). Substrate that has not yet folded rapidly rebinds to GroEL for further folding attempts. 

*Larger substrates fully occupy the limited volume of the GroEL and GroES nanocage, as shown impressively by recent cryo-EManalysis of chaperonin complexes. Encapsulation can accelerate folding up to tenfold over the rate of spontaneous folding. Mutational analysis showed further that the polar residues of the cavity wall are crucial for rapid folding.

*According to molecular dynamics simulations, these polar residues are expected to promote folding by accumulating ordered water molecules in their vicinity, thereby generating a local environment in which a substrate protein is forced to bury exposed hydrophobic residues more effectively. This effect would be significant only with proteins that approach the size limit of the cage, consistent with the finding that folding of smaller substrates of 25–30 kDa is not accelerated.

*Iterative annealing mechanism is also suggested.