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 untaggedtagged 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? MDNTD trans cis

Heterodimeric ATPase assay Determine intra- or intermonomer interactions E33A R376A 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? MDNTD 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-376cis

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-22trans – Val-23trans – Tyr-24cis + trans – Leu-372cis – Leu-374cis E33A catalitically 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-22trans – Val-23trans – Tyr-24cis + trans – Leu-372cis – Leu-374cis

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? Wild type Homomeric double mutations Homomeric single mutations

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? NMC NM NM Hsc82 (WT) 1,20 N599 0,18 N599 / R376A 0,05 Tuncated constructs with and without mutation

NMC NM NM 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

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