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Structural and biochemical characterization of ctTel1, an ATM kinase ortholog from Chaetomium thermophilum
Structural and biochemical characterization of ctTel1, an ATM kinase ortholog from Chaetomium thermophilum
Ataxia-Telangiectasia Mutated (ATM) is an apical signalling kinase that responds to DNA double strand breaks (DSBs). DSBs are the most dangerous form of DNA damage. Therefore cells have evolved multiple pathways to repair such lesions and signal the damage to the cell to organise the DNA damage response (DDR). DSBs are sensed by the Mre11-Rad50-Nbs1 (MRN) complex, which initiates repair via homologous recombination (HR). At the beginning of this process, MRN activates the ATM kinase, which phosphorylates hundreds of substrates, including other DNA repair factors and kinases to orchestrate a cell-wide DNA damage response. ATM belongs to the family of PI3-kinase like kinases (PIKKs). It has a very large HEAT-repeat domain, which plays a role in interacting with other proteins, and a highly conserved kinase domain, showing more homology to PI3-lipid kinases than protein kinases. ATM is normally present in the nucleus as an autoinhibited dimer, but rapidly becomes active upon DNA damage. As a part of its activation mechanism ATM is postulated to monomerise and become autophosphorylated. In order to understand the molecular mechanism of ATM activation better, the aim of this work was to solve the structure of ctTel1, a fungal ATM ortholog from the mould Chaetomium thermophilum, using cryo-electron microscopy (cryo-EM). Due the large size of the protein (650 kDa as a dimer) recombinant expression was not possible. Therefore ctTel1 was puried from endogenous sources. The other aim was to characterise its interaction with MRN and DNA, for which a truncated ctTel1 construct was used which could be obtained in large quantities. Using the truncated construct, the interactions between ATM and Nbs1, and the MRN complex were characterised using pull-downs, analytical gel filtration and fluorescence anisotropy, showing that it forms a stable complex. Furthermore we found that truncated ctTel1 has DNA binding properties, preferring blunt-ended double stranded DNA and displaying a length-dependency. Using cryo-EM we determined the first complete and the highest resolution structure of any ATM kinase to date, with the kinase domain at 2.8 A and an overall structure including the HEAT repeat domain at 3.7 A. This allowed for the building of a complete atomic model, except for a number of flexible loops. The structure reveals a hydrophobic dimer interface, suggesting monomerisation is unlikely to be an aspect of the activation mechanism. In the active site of the kinase the loops required for catalysis are structured, coordinating ATPgS and a Mg2+ ion. This suggests that ATM is in a catalytic proficient state. However, access to the active site is physically blocked by the PIKK regulatory domain (PRD) a regulatory helix. The PRD thus functions as a pseudosubstrate, suggesting that part of the activation mechanism entails its removal in response to allosteric binding of activators. Cancer mutations and ataxia-telangiectasia (A-T) mutations were mapped onto the structure, revealing the elements which are important for the activation. Taken together, the ctTel1 structure reveals the autoinhibitory circuitry and provides the first step towards understanding the activation mechanism of this important kinase.
Not available
Jansma, Marijke
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Jansma, Marijke (2020): Structural and biochemical characterization of ctTel1, an ATM kinase ortholog from Chaetomium thermophilum. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Ataxia-Telangiectasia Mutated (ATM) is an apical signalling kinase that responds to DNA double strand breaks (DSBs). DSBs are the most dangerous form of DNA damage. Therefore cells have evolved multiple pathways to repair such lesions and signal the damage to the cell to organise the DNA damage response (DDR). DSBs are sensed by the Mre11-Rad50-Nbs1 (MRN) complex, which initiates repair via homologous recombination (HR). At the beginning of this process, MRN activates the ATM kinase, which phosphorylates hundreds of substrates, including other DNA repair factors and kinases to orchestrate a cell-wide DNA damage response. ATM belongs to the family of PI3-kinase like kinases (PIKKs). It has a very large HEAT-repeat domain, which plays a role in interacting with other proteins, and a highly conserved kinase domain, showing more homology to PI3-lipid kinases than protein kinases. ATM is normally present in the nucleus as an autoinhibited dimer, but rapidly becomes active upon DNA damage. As a part of its activation mechanism ATM is postulated to monomerise and become autophosphorylated. In order to understand the molecular mechanism of ATM activation better, the aim of this work was to solve the structure of ctTel1, a fungal ATM ortholog from the mould Chaetomium thermophilum, using cryo-electron microscopy (cryo-EM). Due the large size of the protein (650 kDa as a dimer) recombinant expression was not possible. Therefore ctTel1 was puried from endogenous sources. The other aim was to characterise its interaction with MRN and DNA, for which a truncated ctTel1 construct was used which could be obtained in large quantities. Using the truncated construct, the interactions between ATM and Nbs1, and the MRN complex were characterised using pull-downs, analytical gel filtration and fluorescence anisotropy, showing that it forms a stable complex. Furthermore we found that truncated ctTel1 has DNA binding properties, preferring blunt-ended double stranded DNA and displaying a length-dependency. Using cryo-EM we determined the first complete and the highest resolution structure of any ATM kinase to date, with the kinase domain at 2.8 A and an overall structure including the HEAT repeat domain at 3.7 A. This allowed for the building of a complete atomic model, except for a number of flexible loops. The structure reveals a hydrophobic dimer interface, suggesting monomerisation is unlikely to be an aspect of the activation mechanism. In the active site of the kinase the loops required for catalysis are structured, coordinating ATPgS and a Mg2+ ion. This suggests that ATM is in a catalytic proficient state. However, access to the active site is physically blocked by the PIKK regulatory domain (PRD) a regulatory helix. The PRD thus functions as a pseudosubstrate, suggesting that part of the activation mechanism entails its removal in response to allosteric binding of activators. Cancer mutations and ataxia-telangiectasia (A-T) mutations were mapped onto the structure, revealing the elements which are important for the activation. Taken together, the ctTel1 structure reveals the autoinhibitory circuitry and provides the first step towards understanding the activation mechanism of this important kinase.