9 - Molecular Chaperones and Hsp90

Question 1

How do molecular chaperones aid protein folding?

Answer 1

By inhibiting improper attachments and pulling apart illegitimate liasons.
This often takes the form of binding to a part of a folding protein that might otherwise cause aggregation.

Question 2

What catalystic abilities might a molecular chaperone possess?

Answer 2

Proline cis-trans isomerisation
Disulphide bond formation/reduction
Post translational modification

Question 3

Other than aiding protein folding, what services do molecular chaperones provide in the cell?

Answer 3

They act as rescuers to proteins that have already misfolded or aggregated.

Question 4

What property of cellular environments mean that chaperones are very necessary?

Answer 4

Very high protein concentration, favouring aggregation.

Question 5

What is proteostasis?

Answer 5

Cellular control of the amount of total and individual amounts of proteins.

Question 6

What processes can be regulated to control proteostasis?

Answer 6

Translation, folding, activation, degradation.

Question 7

How are molecular chaperones involved in proteostasis?

Answer 7

They can be used to control the rate at which proteins are folded or acitvated.

Question 8

Why are Peptidyl Prolyl Isomerases (PPIs) valuable?

Answer 8

Because proline cis-trans isomerisation is often the rate limiting step in protein folding.

Question 9

Where are Protein Disulphide Isomerases found?

Answer 9

They are localised to the endoplasmic reticulum.

Question 10

What is unique about Hsp70?

Answer 10

It is the only monomeric heat shock protein.

Question 11

How are heat shock proteins named?

Answer 11

By their molecular weight.

Question 12

What catalytic ability do most shock proteins have?

Answer 12

ATPases

Question 13

What do non-catalytic Hsps do?

Answer 13

They only have ‘holding ability’, binding the proteins to increase their activities or acting as a sponge for unfolded proteins to prevent aggregation.

Question 14

How do catalytic Hsps act on misfolded proteins?

Answer 14

Unfold and refold them, or direct them to the proteasome for degradation.

Question 15

What are the basic features of Hsp90?

Answer 15

Inherent ATPase
Dimer
Employs cochaperones
Involved in protein folding by conformation optimisation
Linked to proteasome degradation

Question 16

Why is Hsp90 an important point of study?

Answer 16

Many cyclin dependent kinases are partially or wholly dependent upon its action, so it has implications for cancer research.

Question 17

What is the molecular weight of Hsp90?

Answer 17

Actually closer to 80kDa, the name is a remnant from when its weight was initially misreported due to SDS-PAGE inaccuracies.

Question 18

What is the C-terminal domain of Hsp90 involved in?

Answer 18

Dimerisation

Question 19

What is the N-terminal domain of Hsp90 involved in?

Answer 19

ATPase activity

Question 20

How many domains are there in each Hsp90 monomer?

Answer 20

Three

Question 21

How flexible is Hsp90?

Answer 21

Very flexible when monomeric due to the linker regions between the three domains.

Question 22

Describe the dimerisation domain structure and how they interact.

Answer 22

C-terminal domain comprised of two helices, H4 and H5 that form a four-helix bundle with the same domain on another Hsp90 through strong hydrophobic interaction.

Question 23

Describe the structure of the ATPase domain of Hsp90.

Answer 23

The N-terminal domain is at the opposite end of the molecule to the C-terminal dimerisation domain. Here two helices, H1 and H3 form an ATP binding pocket which Hsp90’s only ß-sheet forms the base of.

Question 24

How is the binding of ATP to Hsp90 made more favourable?

Answer 24

Binding in the Hsp90 pocket stabilises the ATP by resonance stabilisation, delocalising seven of its bonds.

Question 25

How is ATP hydrolysis made even more favourable by Hsp90?

Answer 25

The result is made more stable than the triphosphate form as eight of its bonds are delocalised as opposed to seven in the ATP. Hsp90 also takes advantage of the phosphate charge repulsion.

Question 26

What is the free energy change of Hsp90 ATP hydrolysis?

Answer 26

-7.3kcal per mol

Question 27

What is strange about the way in which ATP binds in Hsp90 compared to most ATPases?

Answer 27

It binds the ATP in its less favourable compact formation, analagous to a cis conformation in stereochemistry as the triphosphate and adenine groups protrude in the same direction from the sugar. This is unfavourable due to the steric clash, so most ATP binding occurs in the extended form.

Question 28

Why did Hsp90 appear at first not to have any ATPase acitivity?

Answer 28

Because when assays were performed using immobilised ATP the immobilisation linker MABA was attached to the adenine C8 atom, which prevents the compact conformation of ATP from occurring as it would in free solution.

Question 29

What three Hsp90 residues have been shown to be instrumental in ATP binding and catalysis?

Answer 29

Asp-79
Arg-32
Glu-33

Question 30

What is the role of Asp-79 in Hsp90, and how was this identified?

Answer 30

Instrumental in ATP binding as it forms a salt bridge with the adenine. Specific mutation of this residue to asparagine caused total loss of binding activity.

Question 31

What are the roles of Arg-32 and Glu-33 in Hsp90, and how was this identified?

Answer 31

Instrumental in catalysis, the Arg polarising the Glu so it can take part in general base hydrolysis. Mutation of the Glu preserved binding but led to total loss of catalytic activity.

Question 32

What is the ATPase activity of Hsp90 dependent upon?

Answer 32

Protein must be in dimerised form, monomers have no activity. This is thought to be the result of interaction between the ATPase domains.

Question 33

What is notable about pyrene as a fluorophore?

Answer 33

Its activity is greatly increased when it is within 6-10A from another pyrene molecule as the pair form an ‘excited dimer’ or eximer.

Question 34

What is used as a non-hydrolysable analogue of ATP?

Answer 34

AMP-PNP

Question 35

How many cysteine residues are found in Hsp90, and where are they?

Answer 35

Only one on each monomer, found on the ATP binding domain.

Question 36

How was it proved that the ATPase domains interact in the Hsp90 dimer?

Answer 36

Using pyrene fluorpohores linked to the two ATPase domain cysteine residues via maleimide. Eximers were shown to be formed and hence the domains move within 6-10A of each other. Hence C-terminal dimerisation necessary for ATPase action as it increases the local concentration of N-terminal ATPase domains.

Question 37

What must happen in order for the N-terminal ends of the Hsp90 monomers to dimerise?

Answer 37

Both must be binding ATP.

Question 38

How does the binding of ATP allow for N-terminal dimerisation of Hsp90?

Answer 38

When ATP binds it allows an ‘ATP lid’ to swing over the hole, exposing a hydrophobic ß-strand. The two ß-strands from either domain then engage in donor strand exchange which links the top of the dimer together.

Question 39

What is required for ATP hydrolysis to occur in Hsp90?

Answer 39

The ATP lid must be closed.

Question 40

How was the structure of the ATP bound Hsp90 dimers solved?

Answer 40

XRD.

Question 41

Why can crystallographic methods not be used for the Hsp90 monomer structure elucidation?

Answer 41

Because as a monomer the linker regions allow for too much flexibility to form ordered crystals.

Question 42

What issues are caused by the size of the Hsp90 monomer when trying to analyse its structure?

Answer 42

It is too large for NMR study and marginally too small for TEM analysis.

Question 43

How has TEM analysis been used to gain approximate structural data for the Hsp90 monomer?

Answer 43

Signal averaging used to improve the scan, and knowledge of the angular relationship of the 2D imaged has allowed for ‘back projection’ to form a 3D model.