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Lidija Berke, Liliana Joachin Rodriguez, Renée de Bruin Supervised by Stefan Rudiger

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

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Lidija Berke, Liliana Joachin Rodriguez, Renée de Bruin Supervised by Stefan Rudiger

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  1. Lidija Berke, Liliana Joachin Rodriguez, Renée de Bruin Supervised by Stefan Rudiger

  2. N M C 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

  3. 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

  4. E33A test 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

  5. 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

  6. ATPase activity homodimers deadweak Does Arg376 (MD) effect ATPase activity of own NTD? Hydrofobic residues coming together in the ATP bound state of Hsp82 (yeast Hsp90) MD NTD Interaction of Arg376 with E33; Cis or trans? trans cis

  7. E33A R376A Heterodimeric ATPase assay Determine intra- or intermonomer interactions WEAK WEAK

  8. ATPase activity homodimers deadweak Does Arg376 (MD) effect ATPase activity of own NTD? Hydrofobic residues coming together in the ATP bound state of Hsp82 (yeast Hsp90) MD NTD Interaction of Arg376 with E33; Cis or trans? trans cis • Cis interaction between Arg376 and E33

  9. Conclusion(part I) • Arg-376cis 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

  10. What is the impact of the residues? ATPase activity homodimers • NTD of one monomer • Thr-22 • Val-23 • Tyr-24 • MD of the other monomer • Leu-372 • Leu-374 • Arg-376 (interaction withthe γ-phosphate of ATP)

  11. What is the impact of the residues? ATPase activity heterodimers • Thr-22 • Val-23 • Tyr-24 • Leu-372 • Leu-374 E33A catalitically dead

  12. What is the impact of the residues? ATPase activity heterodimers • Thr-22 trans • Val-23 trans • Tyr-24 cis + trans • Leu-372 cis • Leu-374 cis E33A catalitically dead

  13. Conclusion (part II) • Thr-22 trans • Val-23 trans • Tyr-24 cis + trans • Leu-372 cis • Leu-374 cis • NTD residues: providing interactions required for hydrolysis of the opposite monomer to proceed • MD residues: both leucinemutations interact in cis to the E33Amutation • suggesting that the role of these residues is to bridge thenetwork between the N-terminal residues on the oppositemonomer and the arginine (R376) that has been shown to interactwiththe γ-phosphate of ATP.

  14. 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

  15. What do we see? X axis: radius of gyration Y axis: probability of the molecule having certain radius of gyration

  16. 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

  17. Conclusion (part III) Inter- and intrasubunit network of hydrophobic residues is directly involved in the stabilization of the closed state. 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: largerhydrophobic group can fit in (despite predictions for the opposite from the crystal structure).

  18. Synergy in ATPase activity between NTD and MD? Wild type Homomeric single mutations Homomeric double mutations

  19. Synergy in ATPase activity between NTD and MD? Wild type Homomeric single mutations Homomeric double mutations Yes! Additive effect with the homomeric double mutation

  20. Are there trans effects in NTD-MD? Tuncated constructs with and without mutation N M C Hsc82 (WT) 1,20 N M N599 0,18 N M N599 / R376A 0,05

  21. Are there trans effects in NTD-MD? Tuncated constructs with and without mutation N M C Hsc82 (WT) 1,20 N M N599 0,18 N M N599 / R376A 0,05 • R376 involved in stabilization of NT - M domain for ATPase activity

  22. N M C 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.

  23. Model Trans interaction: - NTD Residue in one monomer interacting with the other monomer ATP - Interacting with MD of the same monomer Tyr24 -Cis/Trans interaction • Cis interactions: • MD residues in the same monomer

  24. Model • 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

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  26. Additional information

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