Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Abstract

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans^1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy³. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers⁴. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5'-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

Fig. 1. ATPγS–(Mg²⁺)₂ interaction with and regulation of DNA-PKcs.

a, Coulomb potential map of the DNA-PKcs–ATPγS–(Mg²⁺)₂ complex. The expanded view shows ATPγS–(Mg²⁺)₂ binding in the ATP-binding groove. ATPγS (light grey), together with two Mg²⁺ ions (fluorescent green), coordinates the N- and C-lobes, especially the p-loop (plum), catalytic loop (chocolate) and activation loop (azure) of DNA-PKcs (grey). The γ-phosphate group points towards the substrate-binding site. The top left image shows the clear Coulomb potential map for modelling of ATPγS–(Mg²⁺)₂, while the schematic representation below highlights the three units of DNA-PKcs and detailed composition of the head unit. b, Opening of the ATP-binding groove entrance. The residues on both sides of the ATP-binding groove entrance, Trp3805 and Met3929, exhibit an outward rotation that allows docking of the adenine moiety of ATPγS. Apo DNA-PKcs (Protein Data Bank (PDB), 6ZFP) is coloured pink with a mesh surface. c, ATPγS–(Mg²⁺)₂ regulation on the PRD. PRD blocks the substrate-binding site. When ATP binds, PRD is tilted and moves away from its position in the apo structure.

Fig. 2. ATP competitive inhibitors (wortmannin, NU7441, AZD7648 and M3814) and their modes of binding to DNA-PKcs.

a, Inhibitors investigated and their corresponding Coulomb potential maps. b, Binding of wortmannin (green) to the ATP-binding site, where it is covalently modified by the primary amine group of Lys3753. c, Binding of NU7441 (blue) to the ATP-binding site. d, Binding of AZD7648 (purple) to the ATP-binding site. e, Binding of M3814 (cyan) to the ATP-binding site. DNA-PKcs is shown in grey.

Fig. 3. Comparisons of the binding modes among ATPγS–(Mg²⁺)₂ and the four inhibitors.

a, Comparison of the binding modes of ATPγS–(Mg²⁺)₂ and wortmannin in DNA-PKcs. Top, binding conformations of ATPγS–(Mg²⁺)₂ (light grey, ATPγS; fluorescent green, Mg²⁺ ions) and wortmannin (green). Bottom, conformational differences in the binding groove of DNA-PKcs between ATPγS–(Mg²⁺)₂ (grey) and wortmannin (green). b, Comparison of the binding modes of ATPγS–(Mg²⁺)₂ and NU7441 in DNA-PKcs. Top, binding conformations of ATPγS–(Mg²⁺)₂ and NU7441 (blue). Bottom, conformational differences in the binding groove of DNA-PKcs between ATPγS–(Mg²⁺)₂ and NU7441 (blue). c, Comparison of the binding modes of ATPγS–(Mg²⁺)₂ and AZD7648 in DNA-PKcs. Top, binding conformations of ATPγS–(Mg²⁺)₂ and AZD7648 (purple). Bottom, conformational differences at the binding groove of DNA-PKcs between ATPγS–(Mg²⁺)₂ and AZD7648 (purple). d, Comparison of the binding modes of ATPγS–(Mg²⁺)₂ and M3814 in DNA-PKcs. Top, binding conformations of ATPγS–(Mg²⁺)₂ and M3814 (cyan). Bottom, conformational differences at the binding groove of DNA-PKcs between ATPγS–(Mg²⁺)₂ and M3814 (cyan).

Fig. 4. Conformational changes resulting from binding of ATPγS–(Mg²⁺)₂ and competitive inhibitors.

a, The p-loop conformation in apo DNA-PKcs (pink) is fixed by the flanking β-sheets and the electrostatic interaction between Arg3733 and Asp3587. b, Two views, related by rotation of 120^o, of the effect of binding different ligands on the conformation of the p-loop. These conformational changes resemble the movement of a spring leaf. The corresponding movement of the flanking β-sheets transmits a conformational change to the core DNA-PKcs kinase region. Grey, ATPγS; green, wortmannin; blue, NU7441; purple, AZD7648; cyan, M3814. c, Orthogonal views of the p-loop conformations regulating the conformation of the DNA-PKcs kinase region, including the PRD.

Extended Data Fig. 1. Home-made graphene oxide grid for DNA-PKcs/ ATPγS-Mg²⁺₂ complex.

a, TEM image of grid hole with suspended graphene oxide on. b, Cryo-EM micrograph of DNA-PKcs/ ATPγS-Mg²⁺₂ complex. c, Fourier transform of b with the graphene oxide reciprocal lattice circled in white dashed line. The graphene-oxide grid is replicated for the DNA-PKcs/complex studies.

Extended Data Fig. 2. Comparisons between DNA-PKcs and mTOR in complex with ligands.

a, Comparison of ATPγS-Mg²⁺₂ binding in the ligand binding grooves of DNA-PKcs and mTOR (PDB entry: 4JSP). The PIKK regulatory domain (PRD) of mTOR is lifted and removed completely from the substrate-binding site while the PRD of DNA-PKcs still docks there, although it is lifted compared to apo DNA-PKcs. DNA-PKcs/ATPγS-Mg²⁺₂ complex is coloured grey while mTOR/ATPγS-Mg²⁺₂ is coloured orange. PRDs of DNA-PKcs and mTOR are circled in dashed lines. b, Close-up view of the ligand-binding pocket of DNA-PKcs and mTOR. Residues contributing to the pocket are highly conserved between DNA-PKcs and mTOR. The major differences lie in the p-loop, including residue composition and loop conformation. The residues that differ are labeled in the figure. Compared to that of mTOR, the p-loop of DNA-PKcs is positioned more inward and closer to the C-lobe, leaving a narrower channel for the ligands to enter. Moreover, DNA-PKcs p-loop pushes the ligands more toward the C-lobe as the ribose moiety and the phosphate groups of ATPγS-Mg²⁺₂ rotate towards the C-lobe compared to those of mTOR. The adenine moieties overlay well as the deep hydrophobic pockets are highly structurally similar. c, Comparison of mTOR in complex with specific inhibitor Torin2 (PDB code: 4JSX) and pp242 (PDB code: 4JT5) and DNA-PKcs in complex with specific inhibitor AZD7648 and M3814 and close-up view. The major differences are in the p-loop conformations. Moreover, among the few residues that differ, Met3729 of DNA-PKcs is more extended compared to the Ile2163 of mTOR at the equivalent position and acts as the gatekeeper. Moreover, it affects the conformation of DNA-PKcs p-loop when different ligands bind. DNA-PKcs in complex with AZD7648 is coloured purple, DNA-PKcs in complex with M3814 is coloured cyan, mTOR in complex with Torin2 is coloured salmon and mTOR in complex with pp242 is coloured olive.

Extended Data Fig. 3. ATP competitive inhibitors in complex with homologues of DNA-PKcs.

a, Comparison of wortmannin binding on the ATP binding groove of porcine PI3Kγ (PDB entry: 1E7U) and DNA-PKcs. The Lys 833 of porcine PI3Kγ and Lys 3753 of DNA-PKcs react with the furan ring of wortmannin to form a covalent C-N bond and irreversibly inhibit the kinase activity. b, Comparison of AZD7648 binding on ATP binding groove of PI3Kγ (PDB entry: 6T3C) and DNA-PKcs. The 90° rotation of the indole ring from PI3Kγ Trp 812 to DNA-PKcs Trp 3805 provides a better surface and strong stacking effect for the binding of AZD7648. The porcine PI3Kγ/Wortmannin complex is coloured brown. The PI3Kγ/AZD7648 complex is coloured olive.

Extended Data Fig. 4. Ligand binding in the same pocket of DNA-PKcs in higher-order complexes without structurally affecting the assemblies.

a, The Coulomb potential map of DNA-PK holoenzyme in complex with ATPγS-Mg²⁺₂. b, Close-up view of the DNA-PKcs kinase domain in the DNA-PK/ ATPγS-Mg²⁺₂ Coulomb potential map with the model of the kinase domain of the DNA-PKcs/ ATPγS-Mg²⁺₂ complex docked in. c, Close-up view of the Coulomb potential map of NHEJ long-range complex (EMD-23510) and the related kinase domain model (PDB: 7LT3), which includes the ligand binding of ADP. d, EMSA/gel shift assay of Ku70/80, DNA-PKcs and DNA-PKcs inhibitor with DNA (replicated three times). For gel source data, see Supplementary Figure 1.

Extended Data Fig. 5. Hotspot analysis for further structure-guided inhibitor development.

a, Hotspot map of DNA-PKcs ATP binding site contoured at 14. The apolar, donor and acceptor hotspot regions are depicted as yellow, blue and red surfaces with the key contributing residues shown as brown, salmon and blue lines respectively. ATPγS-Mg²⁺₂ is shown in green stick-ball representation with inhibitors wortmannin (grey), NU7441 (pink), AZD7648 (blue) and M3814 (brown) overlaid. b, Surface electrostatic representation of DNA-PKcs in complex with ATPγS (green stick-ball) and inhibitors wortmannin (grey), NU7441 (pink), AZD7648 (blue) and M3814 (yellow) overlaid as lines. AZD7648 and M3814 are seen extending to the edge of the ATP pocket due to unique interactions with residues near the catalytic loop, not seen with the other two inhibitors. Additional lead optimization may involve elaborating compounds to utilize interactions on the catalytic loop (blue arrow) and base of C-lobe (green arrow). Growing compounds further into the groove (yellow arrow) occupied by the activation loop, thereby engaging additional polar and electrostatic contacts, allows extension further away from the ATP binding site.

Extended Data Fig. 6. Cryo-EM data processing of the DNA-PKcs/ligand complexes.

a, Cryo-EM data processing of the DNA-PKcs/ATPγS-Mg²⁺₂ complex. b, Cryo-EM data processing of the DNA-PKcs/wortmannin complex. c, Cryo-EM data processing of the DNA-PKcs/NU7441 complex. d, Cryo-EM data processing of the DNA-PKcs/AZD7648 complex. e, Cryo-EM data processing of the DNA-PKcs/M3814 complex.