Structure of the human ATM kinase and mechanism of Nbs1 binding
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Curated by eLife
Evaluation summary
This manuscript is of broad interest to the DNA-repair and structural biology field. The paper describes new insights into the interaction between ATM and Nsb1, proteins central to repairing DNA double-strand breaks in humans. Overall, the structural cryo-electron microscopy data is solid and the data well analyzed and presented with key claims directly related to and supporting previous known findings.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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 of 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.
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Evaluation summary
This manuscript is of broad interest to the DNA-repair and structural biology field. The paper describes new insights into the interaction between ATM and Nsb1, proteins central to repairing DNA double-strand breaks in humans. Overall, the structural cryo-electron microscopy data is solid and the data well analyzed and presented with key claims directly related to and supporting previous known findings.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Joint Public Review:
ATM is critical for double stranded DNA breaks response, yet, how ATM is activated for this response is poorly defined. Warren and Pavletich have used cryo-electron microscopy to structurally study the interaction between ATM and Nsb1, key proteins which sense DNA-double strand breaks and subsequently activate cell cycle checkpoints and homology-directed repair. They have solved cryo-EM structures of ATM with and without a peptide of Nbs1 bound, both to 2.5 A resolution. In the ATM alone dataset, they were able to subclassify the data to reveal an open and closed state of the protein. These structures are in line with those previously published, but the authors give a detailed account of the dimeric assembly. In the dataset which included the Nsb1 peptide the authors were able to solve a cryo-EM map of ATM with …
Joint Public Review:
ATM is critical for double stranded DNA breaks response, yet, how ATM is activated for this response is poorly defined. Warren and Pavletich have used cryo-electron microscopy to structurally study the interaction between ATM and Nsb1, key proteins which sense DNA-double strand breaks and subsequently activate cell cycle checkpoints and homology-directed repair. They have solved cryo-EM structures of ATM with and without a peptide of Nbs1 bound, both to 2.5 A resolution. In the ATM alone dataset, they were able to subclassify the data to reveal an open and closed state of the protein. These structures are in line with those previously published, but the authors give a detailed account of the dimeric assembly. In the dataset which included the Nsb1 peptide the authors were able to solve a cryo-EM map of ATM with extra density corresponding to 10 residues of the C-terminal FxF/Y motif of Nsb1. They were able to model into this density and locate the peptide within a conserved hydrophobic cleft within the Spiral domain of ATM. They have also used mutagenesis and biochemical assays to assess the importance and specificity of this interaction. Kinetic assays reveal that MRN-dsDNA complex significantly stimulates ATM phosphorylation activity, consistent with the yeast Tel1/MRX-DNA results. Kinetic data also shows that the disruption of Nbs1 peptide binding site does not seem to greatly affect this activity. These data extend the current understanding of how the MRN (Mre11-Rad50-Nbs1) complex may recruit and activate ATM at sites of DNA-double strand breaks, but still leaves the questions of how Rad50 and Mre11 may interact and activate ATM.
Strengths:
This manuscript displays solid well-presented structural and biochemical data. Specifically, the cryo-EM data collection and analysis is complete with detailed workflows describing sub-classifications and data processing to obtain high-resolution maps of ATM with and without the Nsb1 peptide. Even without the novelty of the Nbs1 peptide binding, the map resolution is an improvement upon previously deposited ATM structures. This has improved accuracy and completeness of structural modelling of this protein.Weaknesses:
Using cryo-EM to visualise small peptides binding to a large protein is challenging. Although the cryo-EM data is solid, the overall resolution of the map is 2.5 A but the density corresponding to the peptide is much lower. However, incorporating the mutagenesis biochemical data strengthens the conclusion that the peptide is binding in this region.An overall strength of this paper are the high-quality ATM structures and identification of the Nbs1 FxF/Y peptide binding site in the Spiral domain. The authors also define key structural features of different domains and detail interactions along with the structure analyses. The manuscript could be strengthened by more data or discussion on the hypothesis for the mechanism of ATM activation. Yet, in its current form, the high-quality structure and expert structural analyses, which detail interactions and insights, result in high impact data and report that will be foundational for future studies.
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