Lidija Berke, Liliana Joachin Rodriguez, Renée de Bruin

Supervised by Stefan Rudiger

Hsp90

Chaperone involved in:

• Activation and folding of: - kinases

- steroid hormone receptors- nitric oxide synthase- telomerase

• Mitochondral import

Dimer of 2 monomers consisting of:

• N-terminal domain (NTD); ATP binding and ATP active site• Middle domain (MD); required for ATP hydrolysis• C-terminal domain (CTD); required for dimerization

N

M

C

Hsp90

Conformations of Hsp90

Released energy for:- conformational changes Hsp90?- chaperone function of Hsp90?

Aim: discover key interactions between domains and between the monomers of Hsp90 required for ATPase activity

Heterodimeric ATPase assay

Measure- Importance of a Hsp90 residue

in ATPase activity- Determine intra- or intermonomer

interactions

1) Mix two homodimers with different point mutations

2) Homodimers form heterodimers

• Trans interaction (inter): – WT site on opposite monomer present – -> ATPase activity (50%)

• Cis interaction (intra): – No interaction between two WT-sites– -> no ATPase activity

E33A test

Heterodimerization of yeast Hsp90 (Hsp82) in vitro

• Distinguish untagged Hsp82 homodimer (164 kD) and tagged Hsp82 homodimer (170 kD) with MALDI

• Mix untagged Hsp82 (164 kD) and tagged Hsp82 (170 kD) in a 3:1, 1:1 or 1:3 ratio to detect abundancy of heterodimers

untagged tagged

3:1 1:1 1:3

Does Arg376 (MD) effect ATPase activity of own NTD?

Hydrofobic residues coming

together in the ATP bound state

of Hsp82 (yeast Hsp90)

dead weak

ATPase activity homodimers

Interaction of Arg376 with E33;Cis or trans?

MD NTD

trans

cis

Heterodimeric ATPase assay

Determine intra- or intermonomer

interactions

E33A R376A

WEAK

WEAK

Does Arg376 (MD) effect ATPase activity of own NTD?

Hydrofobic residues coming

together in the ATP bound state

of Hsp82 (yeast Hsp90)

dead weak

ATPase activity homodimers

Interaction of Arg376 with E33;Cis or trans?

MD NTD

trans

cis

• Cis interaction between Arg376 and E33

Conclusion (part I)

Arg-376 (MD) interacting with the γ-phosphate of ATP has a cis-interaction with the NTD active site Glu-33.

There is no catalytic cooperativity between the two NTD’s

– Arg-376 cis

What is the impact of the residues?

• NTD of one monomer– Thr-22– Val-23– Tyr-24

• MD of the other monomer– Leu-372– Leu-374– Arg-376 (interaction with

the γ-phosphate of ATP)

ATPase activity homodimers

What is the impact of the residues?

– Thr-22– Val-23– Tyr-24

– Leu-372– Leu-374

E33A catalitically dead

ATPase activity heterodimers

What is the impact of the residues?

– Thr-22 trans– Val-23 trans– Tyr-24 cis + trans

– Leu-372 cis– Leu-374 cis

E33Acatalitically dead

ATPase activity heterodimers

Conclusion (part II)

• NTD residues: providing interactions required for hydrolysis of the opposite monomer to proceed

• MD residues: both leucine mutations interact in cis to the E33A mutation– suggesting that the role of

these residues is to bridge the network between the N-terminal residues on the opposite monomer and the arginine (R376) that has been shown to interact with the γ-phosphate of ATP.

– Thr-22 trans– Val-23 trans– Tyr-24 cis + trans

– Leu-372 cis– Leu-374 cis

SAXS (Small angle X-ray scattering)

• Large-scale structural data on molecules in solution

• Size and shape of molecules

• Sample exposed to X-rays scattered radiation is registered by detector the scattered intensity I(s) is recorded as a function of momentum transfer s

• Calculations…

• Non-interacting particles with the same size, shape and mass: random positions and orientations in solution

– isotropic intensity distribution proportional to the scattering from a single particle averaged over all orientations

What do we see?

• X axis: radius of gyration

• Y axis: probability of the molecule having certain radius of gyration

The results

• WT: shift in distance distribution (but with AMPPNP not as compact)

• V23A and L374N: addition of AMPPNP has no effect

• T22F: the shift after addition of AMPPNP is comparable with the wild type potein

• The network of interactions is responsible for maintaining the conformational equilibrium.

• Mechanistic understanding for the loss of activity that is observed when these residues are mutated.

• T22F: larger hydrophobic group can fit in (despite predictions for the opposite from the crystal structure).

• Inter- and intrasubunit network of hydrophobic residues is directly involved in the stabilization of the closed state.

Conclusion (part III)

Synergy in ATPase activity between NTD and MD?

Protein Activitypmol of ATP hydrolyzed/pmol 82/min

Hsc82 (WT) 1,2

T22F 3,14V23A 0,25Y24A 0,39

L372D 0,04L374N 0,18R376A 0,18

T22F / R376A 0,75V23A / R376A 0,06Y24A / R376A 0,05L374N / R376A 0,05

Wild type

Homomeric double mutations

Homomeric single mutations

Protein Activitypmol of ATP hydrolyzed/pmol 82/min

Hsc82 (WT) 1,2

T22F 3,14V23A 0,25Y24A 0,39

L372D 0,04L374N 0,18R376A 0,18

T22F / R376A 0,75V23A / R376A 0,06Y24A / R376A 0,05L374N / R376A 0,05

Wild type

Homomeric double mutations

Homomeric single mutations

Yes! Additive effect with the homomeric double mutation

Synergy in ATPase activity between NTD and MD?

Are there trans effects in NTD-MD?

N M C

N M

Protein Activitypmol of ATP hydrolyzed/pmol 82/min

N M

Hsc82 (WT) 1,20

N599 0,18

N599 / R376A 0,05

Tuncated constructs with and without mutation

N M C

N M

Protein Activitypmol of ATP hydrolyzed/pmol 82/min

N M

Hsc82 (WT) 1,20

N599 0,18

N599 / R376A 0,05

-R376 involved in stabilization of NT - M domain for ATPase activity

Tuncated constructs with and without mutation

Are there trans effects in NTD-MD?

Discussion• Research to understand the linkage between

conformational change and ATPase activity in Hsp90 function.

• The residues tested in Hsc82 are conserved, the results apply to all the entire family.

• Novel heterodimeric assay to identify residue interactions:

– Thr22, Val23, Tyr24 (NTD residues) ---- > Trans

– Leu372, Leu374, Arg376 (MD residues) ---- > Cis

– Hydrophobic residues (Thr22) increases ATP hydrolysis and stabilizes the closed conformation

• Close state and dimerization is needed when nucleotide is present to have ATPase activity.

N

M

C

Model

ATP- Interacting with MD of the same monomer

Cis interactions:- MD residues in the same monomer

Trans interaction: - NTD Residue in one monomer interacting with the other monomer

Tyr24-Cis/Trans interaction

NTD dimerization-To stabilize close conformation.-Indirect manner to assist ATPase activity

• Stabilization of the ATP hydrolysis state of Hsp90 requires a very specific NTD-MD conformation.

• Cooperativity between the two monomers.

• Asymmetric model?– Only one ATP site correctly oriented– More structural information required

Model

?

Additional information