Lecture 3 molecular chaperones 2 Flashcards
What are ring chaperones called?
HSP60 known as a chaperonin.
What is the difference between chaperones and chaperonins?
Chaperones is the name for a broad category of proteins that assist in folding e.g., Hsp70, Hsp90 and small heat shock proteins. Chaperonins are large ATP dependent protein complexes that form barrel-shaped structures where unfolded proteins enter and fold in an isolated environment.
Are chaperones ATP dependent like chaperonins?
Not all of them so they cannot be classified as ATP dependent.
What is the structure of Hsp60?
A double donut structure with the Anfinsen cage. This is an enclosed chamber-like structure providing a protected environment where unfolded of misfolded proteins can fold properly.
What are type 1 chaperonins?
7 membered rings of identical 60kDa subunits. Found in the mitochondria and chloroplast. Hsp60 in eukaryotes and GroEL in bacteria.
What are type II chaperonins?
8 membered rings. 60kDa subunits. Found in archaea cytosol and eukaryotic CCT/TRiC.
What does CCT/TRiC stand for?
Chaperonin containing TCP-1/ T-complex protein 1 ring complex. Doesn’t require a co-chaperonin cap like GroES.
How do type 1 chaperonins function?
As double ring complexes that use ATP and a co-chaperonin (GroES) to facilitate proper protein folding.
What is the mechanism of action of GroEL (the most commonly studied type 1 chaperonin)?
Hydrophobic interactions initially capture unfolded proteins, but ATP binding releases them into the hydrophilic folding chamber. ATP hydrolysis drives the cycle and GroES (Hsp10) acts as a cap to create an Anfinsen cage for controlled folding. Alternating ATP binding and hydrolysis in the cis and trans rings regulate the system.
What is the role of GroEL rings compared to GroES caps?
GroEL rings form the double ring structure and GroES cap seals the chamber ensuring a controlled folding environment.
What is the substrate range and mechanism overall of Hsp60 and GroEL?
GroEL and TRiC accept some newly synthesised polypeptides from DNAK (bacteria) or hsp70 (eukaryote). Hsp60 accepts new polypeptides imported from mitochondrion Hsp70.
What is the more detailed mechanism of GroEL?
Substrate binding to GroEL after transfer from Hsp70. Can result in local unfolding. ATP binding then triggers a conformational rearrangement of the GroEL apical domains. This is the followed by the binding of GroES (forms the cis complex) and substrate encapsulation for folding. ADP and GroES dissociate at the same time from the opposite (trans) GroEL ring allowing the release of substrate that had been enclosed in the former cis complex. The new substrate remains encapsulated free to fold for the time required to hydrolyse the 7 ATP molecules in the newly formed cis complex. Binding of ATP and GroES to the trans ring causes the opening of the cis complex.
How do the different domains of GroEL function?
The apical domain interacts with the unfolded polypeptide and GroES. Intermediate domain acts as a hinge, enabling conformational changes. The equatorial domain contains the ATP-binding site driving structural movements. The apical and equatorial domains are in their original positions hydrophobic residues interact with unfolded protein. Intermediate domain causes a rotation of the apical and equatorial domains. Raises the hydrophobic residues away from the cavity. This change reduces the affinity for the bound polypeptide preparing for GroES binding. GroES binds to apical domain capping the chamber. Further rotation of hydrophobic residues away from the cavity. Polypeptide is released into chamber to fold. Lining of the cavity becomes hydrophilic promoting proper folding.
How often do prokaryotes use this assisted folding?
Around 70% polypeptides fold co- and post- translationally in E. coli. 10-15% associate with GroEL.
Which sized polypeptides bind to GroEL?
Small 25kDa polypeptides fold quickly without GroEL. Larger polypeptides require GroEL. The size limit of 55kD for polypeptides in GroEL cavity.
How do eukaryotic chaperones work?
Hsp70 prevents premature folding during protein synthesis. Some domains remain unfolded until the entire protein is made. TRiC takes over post-translationally to ensure proper folding.
What sized polypeptides can attach to TRiC?
Larger multidomain polypeptides often do not fit into TRiC. TRiC at a much lower concentration in cytosol compared to GroEL, fewer polypeptides require TRiC.
What is Hsp90?
A dimer of 90kDa sub-units. The substrates include signaling molecules e.g., kinases, steroid receptors (over-expressed in some cancers). Has a diverse substrate binding mechanism.
What is the Hsp90 cycle?
ATP binds to the N-terminal ATPase domain (ND) of apo-Hsp90 containing the inactive substrate induces a confirmational change and the closure of the ATP lid in the ND. After lid closure the NDs dimerize forming the closed Hsp90 dimer with twisted subunits. ATP hydrolysis. Then the NDs dissociate. The inactive substrate molecule interacts mostly with the middle domain MD and is conformationally activated as Hsp90 proceeds through the ATPase cycle. The cofactors CDC37, HOP, AHA1 and p23 accelerate or slow indicated steps of the cycle.
How do Hsp90s regulate steroid hormone receptors?
Steroid hormone receptor is initially bound to Hsp90 in the cytoplasm. Complex is inactive because Hsp90 prevents the exposure of the nuclear localization signal required for nuclear entry. Steroid hormone binds to its receptor it triggers a confirmational change. Change leads to release of Hsp90 exposing the signal. The activated receptor freed of Hsp90 moves into the nucleus and binds to specific DNA sequences (hormone release elements). This binding activates transcription.
Which proteins do Hsp90 interact with that are linked to cancer?
Epidermal growth factor, human epidermal growth factor receptor 2, mesenchymal-epidermal transition factor, cyclin dependent kinase, protein kinase B, hypoxia induced factor 1 alpha, matix metalloproteinase 2.
How has Hsp90 been implemented in cancer treatments?
Hsp90 ATPase inhibitor (geldanamycin) tested as chemotherapeutic.
What are some examples of other chaperones (Non-ATPases) involved in folding?
In eukaryotes/archaea prefoldin/GimC binds newly synthesised polypeptides and passes them to TRiC-especially actin and tubulin. E. Coli contains trigger factor closely associated with the ribosome, which may replicate the role of DNAK.
What is prefoldin/GimC?
Deliver unfolded proteins to cytoplasmic chaperonins. A heterohexameric complex composed of six subunits so often described as jellyfish like. Composed of two alpha subunits and four beta subunits. It is especially crucial for actin and tubulin folding.
Where does the interaction between prefoldin/Gim C and the substrate occur?
Hydrophobic interactions occur in the interhelix regions allowing unfolded substrates to bind.
What is a trigger factor?
Ribosome-associated chaperone found in prokaryotes, plant chloroplasts and yeast.
What is the structure and abundance of trigger factor?
48kDa and highly abundant with 5-20micromolar.
Where does it bind?
Binds ribosomal L23 protein near the exit tunnel transiently (residence time as the time it stayed being 10seconds). Binds nascent chains via multiple low affinity interactions with inner surface and assists folding by preventing aggregation.
Where does trigger factor occur?
Upstream of DnaJ/K (Hsp70/40) and GroEL/ES (Hsp60/10).
What are the different domains in trigger factor 2?
N-terminal domain to bind to the ribosome, C-terminal domain has the main chaperone activity, structurally homologous to other chaperones like SurA, PPIase domain peptidyl-prolyl isomerase activity which helps catalyze the cis-trans isomerization of proline residues.
What is the function of trigger factor 2?
Associates with the ribosomal exit vestibule, interacting with nascent polypeptides as they emerge. The surface contacts nascent chains to prevent premature misfolding or aggregation. Forms an anfisen cage.
What are trigger factor dynamics in protein biosynthesis?
The trigger factor stays associated to the ribosome for about 10 seconds this is enough time for 200 amino acids to be made. Once the trigger factor detaches from the ribosome it either leaves the nascent chain or stays attached if the nascent chain has hydrophobic regions, sometimes for up to 25s. Long proteins can be covered by multiple TF molecules at the same time.
How does the trigger factor interact with the growing peptide chain?
As the new protein grows, it can touch the entire inner surface of TF. As the chain gets longer, it can follow different paths. 1. Move through TF and become more exposed to the cytosol. 2. Stay protected inside TF, close to the ribosome. TF can hold and shield parts of the protein but may also prevent it from folding too early. Some studies suggest TF can hold entire folded domains or even small proteins but whether it helps folding during translation is still uncertain.
