Structure of the human ATM kinase and mechanism of Nbs1 binding
- Memorial Sloan Kettering Cancer Center, United States
- Memorial Sloan Kettering Cancer Center, Howard Hughes Medical Institute, United States
Abstract
DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and cancer. Central to the sensing of DSBs is the ATM (Ataxia-telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. In response to DSBs, ATM is activated by the MRN (Mre11-Rad50-Nbs1) protein complex through a poorly-understood process that also requires double-stranded DNA. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM, and is required to retain active ATM at sites of DNA damage. Here we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning the N and C lobes along with relieving the blockage of the substrate-binding site. We also show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these structural findings with mutagenesis and biochemical assays.
Data availability
Cryo-EM maps have been deposited in the EMDB under accession codes EMD_25140 and EMD_25141. Coordinates have been deposited in the PDB under accession codes 7SIC and 7SID.
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Structure of the human ATM kinase and mechanism of Nbs1 bindingRCSB Protein Data Bank, 7SIC.
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Structure of the human ATM kinase and mechanism of Nbs1 bindingRCSB Protein Data Bank, 7SID.
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Structure of the human ATM kinase and mechanism of Nbs1 bindingElectron Microscopy Data Bank, EMD-25140.
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Structure of the human ATM kinase and mechanism of Nbs1 bindingElectron Microscopy Data Bank, EMD-25141.
Article and author information
Author details
Nikola P Pavletich
Funding
National Cancer Institute (5F32CA247320)
- Christopher Warren
National Cancer Institute (CA008748)
- Nikola P Pavletich
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Warren & Pavletich
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Structure of the human ATM kinase and mechanism of Nbs1 binding
eLife 11:e74218.
https://doi.org/10.7554/eLife.74218
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Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, local spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-β4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-β4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy.
