Chaperones 2

• Types of ring chaperones:

  • Type 1 - in bacteria and eukaryotic
  • Type 2 - in eukaryotic and cytosol
• Type 1 ring chaperones - 7 membered rings of identical 60 kDa subunits
• Type 2 ring chaperones:

  • 8 membered rings
  • 60 kDa subunits
• Action of type 1 ring chaperones:

  • Ring of the hydrophobic residues lining upper part of cavities binds non native polypeptides from hsp70
  • ATP binding in the cis ring / double ring
  • ATP binding in trans releases GroES and polypeptide
• ATP binding in the double ring causes the movement of hydrophobic residues of chaperonin away from polypeptide which is released into cavity

  Binding of GroES cap results in expansion of cavity to form folding cage with hydrophilic lining
• GroEL (bacteria) and TRiC (eukaryote) accept some newly synthesised polypeptides from DNAK (bacteria) or Hsp70 (eukaryote) - Hsp60 accepts polypeptides newly imported into mitochondrion from Hsp70
• Mechanism of ring chaperones:

  1. Substrate binding to GroEL (may result in local unfolding)
  2. ATP binding triggers conformational rearrangement of the GroEL apical domain
  3. Binding of GroES and substrate encapsulation for folding
  4. ADP and GroES dissociate from the opposite trans GroEL ring - releases the substrate
  5. New substrate remains encapsulated for the time needed to hydrolyse seven ATP molecules in the cis complex
  6. ATP and GroES bind to the trans ring causing the opening of the cis complex
• mechanism of release of polypeptide into the folding cavity:

  1. When ATP binds, apical (A) and equatorial (E) domains rotate around intermediate (I) hinge domain
  2. hydrophobic residues repel from the cavity allowing GroES to bind
  3. Causes further rotation of hydrophobic domains away from the cavity lining - releasing polypeptide to fold in cavity
• Ring chaperones in prokaryotes:

  • Small polypeptides (25 kDa) fold without GroEL
  • Large polypeptides require GroEL
  • Size limit of 55 kDa for polypeptides in GroEL cavity
  • About 10 - 15 % of newly synthesised polypeptides associate with GroEL
• Ring chaperones in eukaryotes:

  • Polypeptides tend to fold in domains co-translationally - assisted by hsc70
  • Non contigous sequences of domains are released to TRiC for post translational folding
  • Larger multi domain polypeptides don't fit into TRiC
• Hsp90 substrates include signalling molecules e.g. kinases, steroid receptors etc
• Hsp90 cycle:

  1. ATP binds to the N terminal ATPase domain of apo hsp90 - induces conformational change and closure of the ATP lid in the n terminal domain
  2. n terminal domain dimerize forming the closed hsp90 dimer with twisted subunits
  3. The stable confirmation is committed for ATP hydrolysis
  4. The n terminal domain dissociates causing the substrate to interact with the middle domain
  5. Substrate is activated
• During the hsp90 cycle, the co factors CDC37, HOP, AHA1, and p23 accelerate or slow steps in the cycle
• Hsp90 and steroid hormones:

  1. Hormone receptor and hsp90 complex formed in cytosol
  2. Hormone binding releases hsp90 and exposes nuclear localization signal
  3. Hormone receptor moves into the nucleus
  4. The active receptor binds to the DNA, activating transcription
• Hsp90 substrates are linked to cancer - hsp90 ATPase inhibitors can be used
• Other non ATPase chaperones involved in folding:

  • Eukaryotes/archaea - GimC binds to newly synthesised polypeptides and passes them to TRiC - especially actin and tubulin
  • E.coli - contains Trigger Factor (TF), closely associated with the ribosome which may replicate the role of DNAK
• GimC is a heterohexamer that delivers proteins to chaperonins
• GimC is a 120 amino acid protein that consists of 2 alpha subunits and 4 beta subunits - substrates bind into the interhelix hydrophobic regions
• GimC acts as a transport molecule to direct target protein and specifally binds to cytoplasmic chaperonin (CCT)
• Trigger factor chaperones are highly abundant 48 kDa protein that binds to ribosomal L23 protein near the ribosomes exit tunnel
• Trigger factor binds nascent chains via multiple low affinity interactions with inner surface and assists folding by preventing aggregation - causes an upstream of DNAK and GroEL / GroES