Molecular Chaperones

MOLECULAR CHAPERONES

Oct 2006 Chaperone lecture notes: 6 slides per page
2 slides per page

EM lecture notes: 6 slides per page (nb: 5.2 MB!)
2 slides per page (nb: 31 pages, 1.8 MB!)

Old lecture notes

Aims and Objectives
Aim: To provide an understanding of the roles, structures and mechanisms of molecular chaperones. To get an overview of the major chaperone families and their modes of interaction with substrates.
Specific objectives: To consider in detail the structural and allosteric basis of chaperonin action.

Definition
A large group of unrelated protein families whose role is to stabilize unfolded proteins, unfold them for translocation across membranes or for degradation, and/ or to assist in their correct folding and assembly.

Properties

Molecular chaperones interact with unfolded or partially folded protein subunits, e.g. nascent chains emerging from the ribosome, or extended chains being translocated across subcellular membranes.
They stabilize non-native conformation and facilitate correct folding of protein subunits.
They do not interact with native proteins, nor do they form part of the final folded structures.
Some chaperones are non-specific, and interact with a wide variety of polypeptide chains, but others are restricted to specific targets.
They often couple ATP binding/hydrolysis to the folding process.
Essential for viability, their expression is often increased by cellular stress.

Main role: They prevent inappropriate association or aggregation of exposed hydrophobic surfaces and direct their substrates into productive folding, transport or degradation pathways.

The structure of an archaebacterial small heat shock protein. It has 24 subunits, each with an immunoglobulin fold, arranged as a hollow shell with holes. It was determined by Kim et al (1998) Nature 394, 595-599, and is a member of the family that includes the eye lens protein alpha-crystallin. [pdb code 1shs]

The structure of the substrate binding domain of the Hsp70 protein DnaK (front and side views, left and center), with a bound peptide (green) in a channel penetrating right through the DnaK domain [pdb code 1dkx]. On the right is the ATPase domain of another member of the Hsp70 family, Hsc70 [pdb code 1kax]. The ATP (space filling) binding site is in a cleft. The ATPase domain structure is homologous to those of actin and hexokinase.

The structure of the chaperonin GroEL (hsp60) Left, a low resolution view of the 14-mer, from the X-ray crystal structure filtered to 25 A resolution. There are 2 contacts (numbered) between the two back-to-back heptameric rings. Right, a single subunit (60 kDa) shown as an alpha-carbon trace. There are three domains, separated by hinge regions (marked H1 and H2). Bound ATP is shown in space filling form, and the yellow residues are hydrophobic sites of substrate (non-native polypeptide) binding. These residues are also required for GroES (hsp10) binding, in addition to the blue residues. The charged residues in the inter-ring contacts are shown in red and blue. The structure was determined by Braig et al (1994) Nature 371, 578-586; Braig et al (1995) Nature Structural Biology 2, 1083-1094.

FAMILIES OF MOLECULAR CHAPERONES

Small heat shock proteins (hsp25) [holders]

protect against cellular stress
prevent aggregation in the lens (cataract)

Hsp60 system (cpn60, GroEL) ATPase [(un)folders]

protein folding

Hsp70 system (DnaK, BiP) ATPase [(un)folders]

stabilization of extended chains
membrane translocation
regulation of the heat shock response

Hsp90 ATPase [holder]

binding and stabilization/ regulation of steroid receptors, protein kinases

Hsp100 (Clp) ATPase [unfolder]

thermotolerance, proteolysis, resolubilization of aggregates

Calnexin, calreticulin

glycoprotein maturation in the ER
quality control

Folding catalysts: PDI, PPI [folders]

Prosequences: alpha-lytic protease, subtilisin (intramolecular chaperones) [folders]

HSP70 AND HSP60 FAMILIES
Location	Chaperone	Roles
HSP70 Family
Prokaryotic cytosol	DnaK cofactors DnaJ, GrpE	Stabilizes newly synthesised polypeptides and preserves folding competence; reactivates heat-denatured proteins; controls heat-shock response
Eukaryotic cytosol	SSA1, SSB1(yeast) Hsc/hsp70, hsp40 (mammalian)	Protein transport across organelle membranes; binds nascent polypeptides; dissociates clathrin from coated vesicles; promotes lysosomal degradation of cytosolic proteins
ER	KAR2, BiP/Grp78	Protein translocation into ER
Mitochondria/ Chloroplasts	SSC1 ctHsp70	Protein translocation into mitochondria; Insertion of light-harvesting complex into thylakoid membrane
HSP60/CHAPERONIN Family *GroE subfamily*
Prokaryotic cytosol	GroEL/ GroES	Protein folding, including elongation factor, RNA polymerase. Required for phage assembly
Mitochondria/ Chloroplasts	Hsp60/10 Cpn60/10	Folding and assembly of imported proteins
TCP-1 subfamily
Archaebacterial cytosol	TF55 Thermosome	Binds heat-denatured proteins and prevents aggregation
Eukaryotic cytosol	TCP-1, CCT, or Tric	Folding of actin and tubulin; folds firefly luciferase in vitro

The GroEL-GroES-ADP complex crystal structure, cut open to reveal the hydrophobic residues (yellow) lining the closed and open cavities (Xu et al, 1997) [pdb code 1aon].

Location of the hydrophobic binding sites (yellow) on the GroEL apical domains in the GroES-bound ring (top) and open ring (bottom) of GroEL. The large twist of the apical domains in the bound ring occludes the binding sites so that substrate proteins, originally bound in the open ring, are ejected from the hydrophobic surface and trapped inside a hydrophilic cavity upon ATP and GroES binding.

Subunit structures of GroEL in the GroES-bound conformation (left), and the thermosome in the closed conformation (right). The chain is coloured from blue (N) to red (C).

Structures of the GroEL-GroES-ADP complex (top) (Xu et al, 1997), [pdb code 1aon], and the closed form of the thermosome (Ditzel et al, 1998) (bottom) [pdb code 1a6d], shown in side view and end view. The domains are coloured red for equatorial, orange for intermediate and yellow for apical. GroES is coloured green. The equatorial domains have detectable sequence homology between GroEL (group I) and the thermosome (group II chaperonins), but the apical domains are not detectably related in sequence. However, aside from the lid extension in the group II apical domain, they have essentially the same fold. GroEL has 7-membered rings, whereas the thermosome has 8 subunits per ring. The group II lid extension takes the place of GroES in closing the chamber, but the enclosed volume is much flatter in the group II oligomer.

The small heat shock protein from Methanococcus and the papD chaperone specific to the papK subunit of the bacterial pilus both have an immunoglobulin fold, and there is either exchange or donation of an edge strand. Unlike the other chaperones discussed here, papD (and related fim proteins) is only a chaperone for its specific substrate, papK. The small hsp dimer is shown above, and the papD-papK complex [pdb code 1pdk] is below, with papD in blue.

The HslUV complex [pdb code 1g3i] is an example of an unfoldase (HslU) coupled to a protease (HslV). This complex contains a hexameric ring of HslU (red and blue) at each end of the central HslV double-ring 14-mer.

The end view of HslU with one subunit coloured in green, to show the inter-subunit contacts. Each subunit has a bound ATP shown in space-filling format. HslU is a member of the Hsp100 family, which forms part of the AAA ATPase superfamily. Many members of this superfamily have unwinding, unfolding or disassembly functions.

A section through the archaeal ribosome complexed with E. coli Trigger Factor (TF). The polypeptide exit tunnel leads to an enclosed space cradled by TF, which is proposed to allow folding of the nascent chain in an environment protected from aggregation and proteolysis.

Molecular Chaperones: References

Reviews

Gething, M.J. & Sambrook, J. (1992) Protein folding in the cell. Nature 355,33-45.

Morimoto, R, Tissieres, A & Georgopoulos, C, Eds. (1994) The biology of heat shock proteins and molecular chaperones. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, USA.

Hartl, FU (1996) Molecular chaperones in cellular protein folding. Nature 381, 571-580.

Bukau & Horwich (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92, 351-366.

Sigler, PB, Xu, Z, Rye, H, Burston, SG, Fenton, WA & Horwich, AL (1998) Structure and function in GroEL-mediated protein folding. Ann. Rev. Biochem. 67, 581-608.

Ranson, N.A., White, H.E. & Saibil, H.R. (1998) Chaperonins. Biochem. J. 333, 233-242.

Saibil, H. (2000) Molecular chaperones: containers and surfaces for folding, stabilising or unfolding proteins. Current Opinion in Struct. Biol. 10, 251-258.

Molecular chaperones - review volume - Advances in Protein Chemistry, vol 59 (2002).

Saibil, H & Ranson, N. (2002) The chaperonin molecular machine, Trends in Biochem. Sci. 27, 627-632.

Sakahira,H, Breuer, P, Hayer-Hartl, Mk, Hartl, FU (2002) Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity. PNAS 99, 16412-16418.

Bukau, B, Weissman, J, Horwich A (2006) Molecular chaperones and protein quality control. Cell 125, 443-451.

Research Papers

Anfinsen, C.B. (1973) Principles that govern the folding of protein chains. Science 181, 223-230.

Zhu, X, Zhao, X, Burkholder, W, Gragerov, A, Ogata, C, Gottesman, M & Hendrickson, W (1996) Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606-1614.

Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D.C., Joachimiak, A., Horwich, A.L. and Sigler, P.B. (1994). The crystal structure of the bacterial chaperonin GroEL at 2.8 Å. Nature 371, 578-586.

Fenton, W.A., Kashi, Y., Furtak, K. & Horwich, A.L. (1994) Residues in chaperonin GroEL required for polypeptide binding and release Nature 371, 614-619.

Roseman, A, Chen, S, White, H, Braig, K & Saibil, H (1996) The chaperonin ATPase cycle: Mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell 87, 241-251.

Buckle, A., Zahn, R. & Fersht, A. (1997) A structural model for GroEL-polypeptide recognition. Proc. Natl. Acad. Sci. USA 94, 3571-3575.

Xu, Z., Horwich, A. & Sigler, P. (1997) The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388, 741-750.

Ditzel et al (1998) The crystal structure of the thermosome. Cell 93, 125-138.

Kim, Kim & Kim (1998) Crystal structure of a small heat shock protein. Nature 394, 595-599.

Rutherford SL, Lindquist S. (1998) Hsp90 as a capacitor for morphological evolution. Nature 396, 336-342.

MC Sousa, CB Trame, H Tsuruta, SM Wilbanks, VS Reddy, and DB McKay (2000) Crystal and Solution Structures of an HslUV Protease-Chaperone Complex.Cell 103, 633-643.

Ferbitz et al (2004) Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431, 590-596.

Structural basis of interdomain communication in the Hsc70 chaperone. Jiang J, Prasad K, Lafer EM, Sousa R. (2005) Mol Cell. 20, 513-524.

Bukau, B, Weissman, J, Horwich A (2006) Molecular chaperones and protein quality control. Cell 125, 443-451.

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Ali MM, Roe SM, Vaughan CK, Meyer P, Panaretou B, Piper PW, Prodromou C, Pearl LH. (2006) Nature 440, 1013-1017.

Structure of an Hsp90-Cdc37-Cdk4 complex. Vaughan, CK, Gohlke, U, Sobott, F, Good, VM, Ali, MMU, Prodromou, C, Robinson, CV, Saibil, HR & Pearl, LH (2006) Mol. Cell 23, 697-707.

Link to Chaperone Group home page at Birkbeck - including movies