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Lidija Berke, Liliana Joachin Rodriguez, Renée de Bruin Supervised by Stefan Rudiger. N M C. Hsp90 Chaperone involved in: Activation and folding of: - kinases - steroid hormone receptors - nitric oxide synthase - telomerase Mitochondral import - PowerPoint PPT Presentation
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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