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.
Reviewing Editor
- Wolf-Dietrich Heyer, University of California, Davis, United States
Version history
- Received: September 25, 2021
- Preprint posted: October 17, 2021 (view preprint)
- Accepted: January 24, 2022
- Accepted Manuscript published: January 25, 2022 (version 1)
- Version of Record published: February 9, 2022 (version 2)
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
Further reading
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The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.
