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Molecular Chaperones

Molecular Chaperones. BIOC 450 23 November, 2009 Jason C. Young. History. 1973: spontaneous folding; sequence and structure 1989: chaperone hypothesis 1991: GroEL pure system 1993: DnaK (Hsp70) pure system; J domain family 1997: Hsp90 ATPase; GroEL mechanism 1998-2000: TPR domains

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Molecular Chaperones

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  1. Molecular Chaperones BIOC 450 23 November, 2009 Jason C. Young

  2. History • 1973: spontaneous folding; sequence and structure • 1989: chaperone hypothesis • 1991: GroEL pure system • 1993: DnaK (Hsp70) pure system; J domain family • 1997: Hsp90 ATPase; GroEL mechanism • 1998-2000: TPR domains • 2001: human genome project • 2001-present: co-chaperone “explosion”

  3. Native State • Native structure is compact and stabilized by multiple hydrophobic contacts • In a folding reaction, the native state has the lowest free energy • protein folding is spontaneous in principle • in practice, impractically slow • Folding intermediates are flexible, less compact, with exposed hydrophobicity cytochrome B562 with Haem Model of folding reaction Daggett and Fersht

  4. Folding vs. Aggregation • Folding intermediates can aggregate with other unfolded polypeptides • Both folding and aggregation depend on hydrophobic interactions • Normal aggregates are structurally disordered • Amyloid fibrils are a special type of aggregate • ordered conformation that is not the native state • pathogenic Dobson (2003) Nature 426, 884-890.

  5. Folding Thermodynamics • each point on curve represents a different polypeptide conformation • each conformation has a different DG • a curve is only one of many possible folding paths unfolded DG intermediate native number of internal contacts compactness coverage of hydrophobicity

  6. many different unfolded and partially folded states different folding pathways lead to one native state intermediates can persist in local free energy minima “kinetically trapped”, requires energy to escape minimum Folding Landscape Folding Landscape Local Minimum Native State: Global Minimum Dobson (2003) Nature 426, 884-890

  7. Folding / Aggregation Landscape • multiple intermolecular contacts in aggregates can make them more stable than individual native state • amyloid aggregates are the most stable

  8. How Do Chaperones Help Folding? • In theory, chaperones could: • prevent aggregation • prevent off-pathway intermediates • unfold off-pathway intermediates to re-start folding • increase on-pathway rates off-pathway intermediate (kinetically trapped) X X + + + X X on-pathway intermediate leads to native state

  9. Chaperonins GroES cofactor Apical domain ATPase domain GroEL E. coli GroEL 14 identical subunits 2 rings of 7 subunits ATPase domain Apical domain down: no GroES bound, hydrophobicity exposed up: GroES bound, polar surface exposed apical domain ATPase domain Bukau & Horwich (1998) Cell 92, 351-366

  10. GroEL Folding Cycle • binding to apical domains • stretching – partial unfolding • confinement • prevents aggregation • favours compact over extended conformations Hartl & Hayer-Hartl (2009) Nature Struc Mol Biol 16, 574-581

  11. Hsp70 Proteins • “Polypeptide Clips” – monomers which bind short, extended hydrophobic regions of substrate • ATPase domain allosterically controls substrate binding domain • substrate binding domain opens and closes DnaK (E. coli) Substrate binding domain (SBD) ATPase domain (NBD) extended conformation

  12. Hsp70 Proteins • human Hsc70 is constitutively expressed, Hsp70 is inducible • also have forms in human mitochondria, ER lumen • co-chaperones: J domain proteins, and nucleotide exchange factors (NEF)

  13. Hsp70 States • biochemical, biophysical, structural data • ATP-bound: • NBD and SBD locked together • SBD pulled open • ADP-bound: • NBD and SBD separate • SBD shut, substrate binding • two-state mechanism conserved in all Hsp70 proteins

  14. J Domain Co-chaperones • J domains bind Hsp70s and are necessary to stimulate ATP hydrolysis • Type 1 DnaJ proteins have J domains and substrate binding domains • Some proteins have J domains but do not bind substrate • activate Hsc70 for specialized functions substrate binding yeast Ydj1 (Type 1) J domain dimerization Type 1 co-chaperones Other J domain co-chaperones Ramos et al. J Mol Biol (2008) 383, 155-166

  15. Hsp70 Cycle • DnaJ binds substrate • J domain stimulates ATP hydrolysis by Hsp70 • substrate binding by Hsp70 in ADP state • NEFs cause ATP re-binding and release of substrate Hartl & Hayer-Hartl (2009) Nature Struc Mol Biol 16, 574-581

  16. Human Hsc70 • pure protein system: Hsc70, unfolded luciferase • DnaJA2 essential • NEF Hsp110 not essential, but enhances • model to study mechanism of Hsc70 and DnaJ

  17. How Does Hsc70 Help Folding? • In theory, chaperones could: • prevent aggregation • prevent off-pathway intermediates • unfold off-pathway intermediates to re-start folding • increase on-pathway rates • which of these apply? X X + + + X X

  18. Aggregation Prevention • Hsc70 binding to hydrophobic intermediate prevents interactions with other unfolded polypeptide • evidence: expect an optimal rate of binding and release • NEFs can be adjusted to an optimal ratio

  19. Confinement • GroEL encloses substrate entirely in cavity • prevents larger intermediates from forming • mutations on inside change folding activity • monomeric Hsc70 not large enough • Hsc70 and DnaJ could bind at once * * GroEL * mutations inside cavity

  20. Unfolding / Annealing • GroEL: substrate binding domains move apart before enclosing • fluorescence spectroscopy • substrate becomes less compact during GroEL binding, then more compact after enclosure • partial unfolding • not always productive • Hsc70 and DnaJ could bind at once

  21. Hsc70 and DnaJ Coordination • human DnaJA2 and A1 are very similar • J domains and substrate binding domains are functional • DJA2 supports folding in model, DJA1 does not; chimera inactive • domain coordination must be different • DJA1 function in cells – different folding challenge?

  22. DnaJ Transfer to Hsc70 • binding of substrate by both DnaJ and Hsc70 during transfer • double binding could change substrate conformation • compress substrate (confinement) • pull apart (partial unfolding)

  23. Increase Folding Rate • analogy to enzyme: reduction of free energy barrier on pathway • barrier could be rotational freedom around peptide bond • Hsc70 binding could promote rotation • evidence: synthetic peptide isomerization • role of DnaJ + +

  24. The Hsc70-Hsp90 Multichaperone System (NEF) Hsc70 and Hsp90: ATP-dependent “folding machines” TPR domains: adaptors that recognize Hsc70 or Hsp90 FKBP52: peptidyl-prolyl isomerase CHIP: E3 ubiquitin ligase Young et al. (2004) Nature Reviews Mol. Cell Biol. 5, 781-791.

  25. The Chaperone-Tom70 Pathway Bag NB GA ATP Hsp90 Hsc70 preprotein Tom70 Tom40 Import Pore Tim9/10 novobiocin, geldanamycin: Hsp90 inhibitors Bag: Hsc70 NEF Young et al. (2003) Cell 112, 41-50 Fan et al. (2006) J. Biol. Chem. 281, 33313-33324.

  26. Chaperone-Tom70 Mechanisms • precursors are inner membrane proteins • Hsc70 could: • prevent aggregation • prevent off-pathway intermediates • unfold off-pathway intermediates to re-start folding • increase on-pathway rates – no • coordinate with Tom70 for transport across membrane? off-pathway intermediate (kinetically trapped) X Tom70 Tom40

  27. Questions Hsp90 DnaJ Hsc70

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