Ataxia-telangiectasia mutated (ATM) is a large protein kinase with a central role in the cellular response to DNA double-strand breaks (DSBs) and related genotoxic stress (Shiloh, 2006). Mutations in ATM are responsible for Ataxia-telangiectasia (AT), which is a rare, autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, sensitivity to radiation, and cancer susceptibility (Savitsky et al., 1995).

ATM, which belongs to the phosphatidylinositol 3-kinase-like protein kinase (PIKK) family (Savitsky et al., 1995), is essential for the sensing of DSBs during the cell cycle. It functions in association with the MRN protein complex consisting of the Mre11, Rad50, and Nbs1 proteins (Syed and Tainer, 2018). MRN contributes to the localization of ATM to DSBs, and together with double-stranded DNA (dsDNA), it activates ATM as a protein kinase. ATM then phosphorylates a wide range of downstream effector proteins, such as p53, Chk2, Brca2, and H2A.X, leading to the activation of cell cycle checkpoints and homology-directed repair (HDR) (Dinkelmann et al., 2009; Shibata et al., 2014; Paull, 2015; Uziel et al., 2003; Lee and Paull, 2004; Lee and Paull, 2005). Mutations in all three members of the MRN complex cause disorders that are phenotypically similar to AT (Stewart et al., 1999; Digweed and Sperling, 2004; Ragamin et al., 2020).

The MRN complex rapidly associates with DNA ends upon DSB formation, and it is a known component of ionizing radiation-induced foci (IRIF) (Lukas et al., 2004; Haince et al., 2008). The Mre11 subunit has endonuclease and exonuclease activities that are implicated in DNA end processing (Hoa et al., 2016; Myler et al., 2017), although nuclease-inactive mutants in yeast have only mild defects in responding to ionizing radiation (IR) (Symington and Gautier, 2011). Rad50 is a member of the structural maintenance of chromatin (SMC) family of proteins. It contains an ATPase domain, a long antiparallel coiled coil, and a zinc hook domain that is thought to tether multiple MRN complexes (de Jager et al., 2001; Hopfner et al., 2002; Moreno-Herrero et al., 2005). Nbs1 contains N-terminal FHA and BRCT domains that are required for association with phosphorylated proteins such as H2A.X, CtBP-interacting protein (CtIP), and mediator of DNA damage checkpoint protein 1 (Mdc1) at IRIF (Kobayashi et al., 2002; Spycher et al., 2008). While the C-terminal half of Nbs1 is largely disordered Williams et al., 2009 it contains short sequence motifs for binding to Mre11 and ATM (Schiller et al., 2012; You et al., 2005).

The activation of ATM by MRN is incompletely understood. The yeast homolog of ATM, Tel1, can associate with each of the individual subunits of the corresponding Mre11-Rad50-Xrs2 complex (Hailemariam et al., 2019). In the yeast system, Xrs2 is required for Tel1 recruitment to DSBs in vivo (Oh et al., 2016; Oh et al., 2018), but in vitro Tel1 activation appears more dependent on Rad50, as either Rad50-Mre11 or Rad50-Xrs2 but not Mre11-Xrs2 can partially activate Tel1 in the presence of DNA (Hailemariam et al., 2019). The ATM-binding segment of Nbs1 has been mapped to a sequence motif termed FxF/Y (residues 740–749) at the C-terminus (You et al., 2005). Those of Mre11 and Rad50 have not yet been identified. The Nbs1 FxF/Y motif is necessary for ATM recruitment to MRN-bound DSBs and for ATM activation in Xenopus extracts You et al., 2005 and human cell lines (Falck et al., 2005). However, FxF/Y deletion affects only a subset of the checkpoint functions, with non-uniform effects on downstream substrate phosphorylation (Falck et al., 2005). In addition, fibroblasts from mice harboring an Nbs1 C-terminal deletion (Nbs1^∆C/∆C) do not display overt sensitivity to IR, although they exhibit defective intra-S phase checkpoint activation, apoptosis induction, and phosphorylation of a subset of ATM targets (Stracker et al., 2007). Taken together, these results suggest that the ATM-Nbs1 interaction is not strictly required to initiate ATM activation, but it is likely necessary to stabilize activated ATM at sites of DNA damage and is critical for certain ATM-mediated checkpoint functions. This may reflect the ability of the remaining MRN subunits to also interact with ATM, or additional factors within IRIFs may aid in the recruitment of ATM in vivo to partially compensate for the loss of the Nbs1 C-terminus.

Along with ATM, the PIKK family also includes ATR, DNA-PKcs, mTOR, SMG1, and TRRAP (Imseng et al., 2018). These mammalian PIKKs coordinate diverse cellular processes, with ATR involved in the response to collapsed replication forks, DNA-PKcs in non-homologous end joining (NHEJ), mTOR in metabolism and cellular homeostasis, SMG1 in nonsense-mediated mRNA decay, and the catalytically inactive TRRAP in scaffolding chromatin remodeling complexes (Lovejoy and Cortez, 2009; Baretić and Williams, 2014). Like ATM, these PIKK kinases are switched on by binding to their cognate activating proteins or protein complexes. The PIKKs share sequence homology across their C-terminal portion that consists of the ~700-residue FAT domain (residues 1899–2613 of ATM) and subsequent ~400-residue kinase domain (KD; residues 2614–3056 of ATM). Their N-terminal regions are divergent, and they adopt α−α solenoid structures typically consisting of two or more helical-repeat domains.

The cryo-EM structure of human ATM, based on a reconstruction of 4.4 Å to 5.7 Å resolution, showed that it forms a dimer through interactions between the FAT domains and also between the FAT and kinase domains (Baretić et al., 2017). The FAT-KD interactions involve a region of the KD, termed the PRD (PIKK Regulatory Domain), that sequesters the putative polypeptide substrate-binding site (Langer et al., 2020), as deduced from comparisons to canonical protein kinases (Brown et al., 1999). This arrangement was thus suggested to maintain ATM in an inactive state by inhibiting substrate binding (Baretić et al., 2017). The N-terminal α−α solenoids are uninvolved in dimerization (Baretić et al., 2017). A subsequent cryo-EM analysis reported a monomeric form at 7.8 Å resolution, in addition to the canonical dimer at 4.3 Å (Xiao et al., 2019). The Tel1 homolog has been amenable to higher resolution structure determination (Xin et al., 2019), with recent structures of the Chaetomium thermophilum and Sacharomyces cerevisiae Tel1 reported at overall resolutions of 3.7 Å and 3.9 Å, respectively (Jansma et al., 2020; Yates et al., 2020). These Tel1 structures exhibited similar dimerization interfaces as ATM, including the region that blocks part of the putative substrate-binding site. Based on the higher resolutions of the Tel1 reconstructions, it was suggested that the kinase active site residues are in a catalytically competent organization, with the implication that the inaccessibility of the substrate-binding site is the primary mechanism of keeping ATM inactive. While Tel1 and ATM share a similar structural organization, the N-terminal ~1,800 residues preceding the FAT domain do not have detectable sequence homology.

Here, we report the cryo-EM structure of human ATM as well as the structure of ATM bound to a peptide containing the Nbs1 FxF/Y motif, both at an overall resolution of 2.5 Å. The organization of residues in the ATM catalytic cleft are very similar to those of the inactive states of mTOR and DNA-PKcs, the two PIKKs for which high-resolution structures have been reported for both the inactive and active states (Yang et al., 2013; Yang et al., 2017; Sibanda et al., 2017; Chen et al., 2021b; Chen et al., 2021a; Chaplin et al., 2021). Notably, ATM does not exhibit the conformational change that is characteristic of the active states of mTOR and DNA-PKcs kinase domains (Yang et al., 2017; Sibanda et al., 2017; Chen et al., 2021b). Together with biochemical data, this suggests that activation of ATM by MRN may involve a conformational change that, in addition to relieving the partial blockage of the substrate-binding site, also realigns catalytic residues.

Our cryo-EM structure of human ATM shows an overall symmetric dimer, albeit with the Pincer and Spiral domains exhibiting some mobility arising from conformational flexibility within the Pincer domain. Nevertheless, this conformational flexibility does not translate to any noticeable structural rearrangements within the FATKD segment, nor does it break the inherent symmetry of this segment (Figure 1—figure supplement 6). Previous cryo-EM studies of human ATM and yeast Tel1 have observed asymmetric dimers and monomers, the latter of which was shown to have increased catalytic activity (Baretić et al., 2017; Xiao et al., 2019). In our purifications and subsequent cryo-EM structure determination, we failed to find any particles representing asymmetric dimers or monomers, also consistent with the most recent cryo-EM structures of Tel1 (Jansma et al., 2020; Yates et al., 2020). While it is not clear how the asymmetric dimers and monomers arise, it is conceivable that expression and purification conditions may play a role. Detergents have previously been demonstrated to disrupt the ATM dimeric structure and increase ATM kinase activity independent of MRN (Lee and Paull, 2006). We note that the report of active monomer preparations demonstrated only an approximately 10-fold increase in catalytic activity relative to the inactive dimeric state (Xiao et al., 2019), whereas our quantitative kinase assays show a ~100 fold activation upon MRN + long dsDNA addition (Figure 5—figure supplement 1H).

As noted in previous ATM and Tel1 structures (Baretić et al., 2017; Xin et al., 2019; Jansma et al., 2020; Yates et al., 2020), the apparent reason for the inhibited state of the ATM dimer is the kα9b helix packing against the putative substrate-binding site and inserting a glutamine side chain into the activation loop, at a site where a glutamine side chain from the substrate would bind (Figure 3—figure supplement 1; Langer et al., 2020). As the kα9b helix packs with the TRD3 coiled coil of the other protomer in the dimer, an interaction that possibly stabilizes the kα9b conformation, the TRD3 coiled coil was proposed to couple ATM dimerization to autoinhibition (Baretić et al., 2017; Xin et al., 2019; Jansma et al., 2020; Yates et al., 2020).

The kα9b helix is part of the PRD domain, which was first described based on a yeast two-hybrid screen to identify the part of the ATR PIKK that interacts with its activator TopBP1 (Mordes et al., 2008). The screen led to a minimal ATR fragment that encompasses kα9b and kα10. While scanning mutagenesis identified an ATR kα10 residue that disrupts TopBP1-mediated activation (Mordes et al., 2008) it is not clear what role kα10 plays in PIKK activation. The mutation (K2589E) maps to a surface residue uninvolved in any interactions in the inactive ATR structure (Rao et al., 2018), and the corresponding ATM residue (Glu3007) is similarly surface-exposed and devoid of any interactions. In addition, the kα10 helix is an integral part of the PIKK C lobe structure, and it is structurally invariant among inactive and active PIKK structures. It is thus unlikely that kα10 has a significant role in conformationally regulating the kinase. In ATM, the kα9b and kα10 helices are connected by a 27-residue loop (residues 2975–3000) that is not conserved across species and is not visible in our map (Figure 1—figure supplement 4). An unstructured loop intervening the kα9b and kα10 helices is present in most PIKK structures reported to date, including a >1000 residue insertion in Smg1³⁵.

In structures of inactive DNA-PKcs, kα9b and subsequent kα9c, a helix unique to DNA-PKcs, also pack against the substrate-binding site and occlude it (Sibanda et al., 2017; Chen et al., 2021b). However, even though DNA-PKcs is glutamine directed as ATM, it does not have the equivalent of the Gln2971-activation loop interaction of ATM. Nevertheless, upon DNA-PKcs activation, the pair of kα9b and kα9c helices move to an alternate packing position on the C lobe and fully expose the substrate-binding site (Chen et al., 2021b). The movement of kα9b-kα9c is likely due entirely to the realignment of the N and C lobes of DNA-PKcs, as this segment only interacts with the kinase domain (DNA-PKcs does not have the equivalent of the ATM TRD3 coiled coil packing with and possibly stabilizing kα9b across a dimer interface). By contrast, in mTOR, which is not glutamine directed and instead prefers a hydrophobic residue at the P₊₁ position, the kα9b helix does not block the putative substrate-binding site, nor does it change conformation on activation (Yang et al., 2017). Thus, while kα9b may play a role in the autoinhibition of ATM and a number of other PIKKs, this is not a conserved feature of the PIKK family.

Rather, several lines of evidence point to the realignment of the N and C lobes of the kinase domain as the key event in the activation of ATM. As discussed above, the relative orientation of the N and C lobes of ATM is remarkably similar to those of the inactive-state mTOR and DNA-PKcs structures, and distinct from those of the active-state counterparts, which cluster together (Figure 3F). The proper alignment of the N and C lobes is central to kinase activation, as residues critical for substrate binding and catalysis are distributed across the two lobes (Yang et al., 2017).

The mechanism of a PIKK activation was initially established by comparing the inactive and active states of the mTOR holoenzyme (Yang et al., 2017). That work pointed to the FAT domain being the key autoinhibitory element that keeps the N and C lobes and their active site residues in an unproductive configuration (Yang et al., 2017). Binding of the mTOR activator, the small GTPase Rheb, causes a motion of the N-heat solenoid (comparable to the ATM Spiral in its location in the primary sequence but not in its structure). The motion of the N-heat solenoid pulls and twists the FAT domain on which it is anchored, causing it to undergo an extensive conformational change. This shifts the HRD of the FAT domain away from the N lobe, allowing the N lobe to relax to a productive configuration relative to the C lobe. The inhibitory effect of the FAT domain is supported by activating mTOR mutations, which map to residues that couple the N and C lobes and the FAT domain (Yang et al., 2017).

The recent cryo-EM structures of activated DNA-PKcs recapitulate the FAT conformational change observed in mTOR (Chen et al., 2021b). DNA-PKcs activation is triggered by the N-heat solenoid binding to the dsDNA end, stabilized by the Ku70-Ku80 complex. The Ku complex interacts with DNA and the N- and M-heat DNA-PKcs domains, but it also utilizes two flexibly linked elements for additional contacts to DNA-PKcs. These may reflect an initial recruitment interaction, a role that the Nbs1 Nc28 peptide may also be involved in (Figure 4—figure supplement 2E). On DNA-end binding, the DNA-PKcs N-heat solenoid moves relative to M-heat. As with mTOR, N-heat is anchored on the FAT domain, and its motion allosterically induces a conformational change in the FAT domain that realigns the N and C lobes.

In both mTOR and DNA-PKcs, the N-heat solenoids that move are anchored on the FAT TRD2-TRD3 interface, at essentially the same location. And, even though their overall N-heat solenoids are structurally distinct, they both use 4-helix bundle followed by a loop to interact with their respective FAT domains. Our ATM structure reveals a remarkably similar interface between the FAT domain and the Pincer (equivalent to M-heat of mTOR and DNA-PKcs) from the same protomer. The similarities include an identical TRD2-TRD3 site of the FAT domain and a 4-helix bundle and loop element from the Pincer that binds to it, in essentially the same configuration as those of mTOR and DNA-PKcs (Figure 5—figure supplement 2A). Based on this structural conservation and the high level of FAT conservation across the PIKK family, it is conceivable that ATM activation also involves this same mechanistic step, with the activator MRN-dsDNA complex triggering the Pincer to pull on and twist the FAT domain to realign the N and C lobes (Figure 5—figure supplement 2B).

If movement of the Pincer domain is on the pathway of ATM activation, we presume dsDNA and MRN binding will involve portions of ATM beyond the Spiral that binds to Nc28 and which has been proposed to have site(s) of dsDNA binding Jansma et al., 2020. The Spiral has a single, isolated interface with the Pincer domain, and it makes no other interactions to the remainder of the ATM dimer. As such, binding events that are limited to the Spiral would be unlikely to cause a movement or conformational change at the Pincer domain. With mTOR, the activator Rheb bridges one end of the N-heat solenoid to portions of mTOR that are invariant during activation, thus providing a pivot point for the movement of the other N-heat end anchored on the FAT domain (Yang et al., 2017). While DNA-PKcs is more complex, the extensive interactions of N-heat and M-heat along their solenoids appear to similarly serve as pivot point(s) for the movement of N-heat at its FAT anchor (Chen et al., 2021b). It is thus likely that dsDNA-MRN either engage additional ATM domains beyond the Spiral, or they bridge the two Spiral domains of the dimer in a manner that changes their relative orientation, with the resulting change propagating to the Pincer domains (Figure 5—figure supplement 2B).

Because the FAT domain plays a central role in the dimerization interface of the inactive ATM dimer, a conformational change in the FAT domain may well affect the relative arrangement of the two ATM protomers. This may disrupt the packing of kα9b with the TRD3 coiled coil from the second protomer, allowing the movement of kα9b to expose the P₊₁ substrate-binding site. Our steady state kinetic analysis showed that MRN + dsDNA increases the k_cat value of ATM phosphorylating a p53 substrate by over two orders of magnitude without affecting the K_M value (Figure 5—figure supplement 1C to H). This mirrors the steady-state kinetic constants of mTOR activation (Yang et al., 2017). However, activation having no effect on the K_M value was unexpected, given that the presumed binding site for the P₊₁ position of the substrate is blocked in the structure. This may be analogous to findings with the canonical Cdk2-CyclinA kinase, which is activated by phosphorylation on its activation loop. Even though phosphorylation reorganizes the substrate-binding site on the Cdk2 activation loop, the majority of the increase in catalytic efficiency is reflected in an increased rate of phosphoryl group transfer step (Hagopian et al., 2001). Based on the model proposed for Cdk2 (Hagopian et al., 2001), it is possible that the blocked P₊₁ position in inactive ATM allows substrate binding, but with the phospho-acceptor group in an unfavorable position or orientation that reduces the rate of phosphoryl group transfer.

In conclusion, our structural data supports the model that activation of the ATM kinase domain may be mechanistically analogous to the activation of mTOR and DNA-PKcs. While it is not known how Rad50 and Mre11 interact with ATM, we find that the Nbs1 FxF/Y motif binds to the ATM Spiral domain, and the Spiral domain of Tel1 was shown to bind to dsDNA independently of MRN (Jansma et al., 2020). Thus, it is possible that these Spiral interactions cooperate with additional contacts by MRN to trigger a motion of the Pincer domain. This would then result in a conformational change in the FAT domain that not only realigns the N and C lobes of the kinase domain but also relieves the blockage of the substrate-binding site.

The refined dimer structures and corresponding cryo-EM maps (including consensus, symmetry expanded, focused, and composite maps) for unbound and Nc28-bound ATM have been deposited within the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) under accession codes 7SIC and EMD-25140 (unbound) and 7SID and EMD-25141 (Nc28-bound).

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Author details

1. Christopher Warren

   Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0320-7248

2. Nikola P Pavletich

   1. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
   2. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   Conceptualization, Funding acquisition, Supervision, Validation, Writing – review and editing

   For correspondence

   pavletin@mskcc.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6039-3956

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