Cryo-EM structure of human DNA-PK holoenzyme

Abstract

DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase complex composed of a catalytic subunit (DNA-PKcs) and KU70/80 heterodimer bound to DNA. DNA-PK holoenzyme plays a critical role in non-homologous end joining (NHEJ), the major DNA repair pathway. Here, we determined cryo-electron microscopy structure of human DNA-PK holoenzyme at 6.6 Å resolution. In the complex structure, DNA-PKcs, KU70, KU80 and DNA duplex form a 650-kDa heterotetramer with 1:1:1:1 stoichiometry. The N-terminal α-solenoid (∼2 800 residues) of DNA-PKcs adopts a double-ring fold and connects the catalytic core domain of DNA-PKcs and KU70/80-DNA. DNA-PKcs and KU70/80 together form a DNA-binding tunnel, which cradles ∼30-bp DNA and prevents sliding inward of DNA-PKcs along with DNA duplex, suggesting a mechanism by which the broken DNA end is protected from unnecessary processing. Structural and biochemical analyses indicate that KU70/80 and DNA coordinately induce conformational changes of DNA-PKcs and allosterically stimulate its kinase activity. We propose a model for activation of DNA-PKcs in which allosteric signals are generated upon DNA-PK holoenzyme formation and transmitted to the kinase domain through N-terminal HEAT repeats and FAT domain of DNA-PKcs. Our studies suggest a mechanism for recognition and protection of broken DNA ends and provide a structural basis for understanding the activation of DNA-PKcs and DNA-PK-mediated NHEJ pathway.

Figure 1

Overall structure of the DNA-PK complex. (A) Color-coded domain architecture of human DNA-PKcs, KU70 and KU80. The same color scheme is used in all of the structure figures if not otherwise specified. The secondary structure of the biotinylated DNA is shown with the free and blocked DNA ends as indicated. The bases that were not built into the structural model are colored in gray. The inter- and intra-molecular contacts are shown as connected lines. Three major contacts (Interface-1 to Interface-3) between DNA-PKcs and KU70/80 are indicated. (B-D) Ribbon representations of the DNA-PK complex in three different views. The Core^DNA-PKcs (composed of the FAT, KD and FATC) is indicated with dashed rectangle. The DNA is colored in yellow. The bases of DNA are shown in stick representations. FAT, FRAP, ATM, TRRAP domain; FATC, FAT C-terminal domain; KD, kinase domain; M-HEAT, middle HEAT repeats; N-HEAT, N-terminal HEAT repeats.

Figure 2

Structure of DNA-PKcs in the DNA-PK complex. (A-C) Ribbon representations of the DNA-PKcs in three different views. The domains are indicated and colored as in Figure 1A. The sizes of the large ring (M-HEAT) and the small ring (N-HEAT and part of FAT) are indicated. Note that the catalytic cavity faces outward (A) and the M-HEAT primarily makes contacts with N-HEAT on repeats HEAT-9 to HEAT-17 (B).

Figure 3

Intermolecular interactions within the DNA-PK complex. (A) Close-up views of intermolecular interactions within DNA-PK in two different views. Ribbon representations are shown with three major contact interfaces indicated. Note that a helix (KU80ctα2) binds to DNA-PKcs and obscures the free DNA end. The DNA-binding tunnel forces DNA duplex to undergo a 30° kink. (B) Close-up views of the electrostatic potential surface of DNA-PKcs in two different views. The inner surface of DNA-binding tunnel within DNA-PKcs is rich in positively-charged (blue) residues. KU80ctα2 is omitted for clarity.

Figure 4

Kinase activity assays of DNA-PKcs. Phosphorylation of p53 (A), HSP90a (B), or MYC (C) by DNA-PKcs in the presence of KU70/80 and/or increasing amounts of DNA. Y-shaped DNA employed in the holoenzyme assembly or sonicated calf thymus DNA is used as indicated. Purified proteins used for the assays are indicated in Supplementary information, Figure S1. Protein and DNA concentrations used in the assay are indicated. Substrate phosphorylation and/or DNA-PKcs autophosphorylation were detected by antibody (A) or autoradiography (B-C).

Figure 5

Mechanism for activation of DNA-PKcs. (A-C) Superimposition of the DNA-PKcs in the apo form (PDB: 5LUQ, molecule B) and the holo form shown in three different views. Transition from the apo form (colored in gray) to the holo form (colored as in Figure 1A) indicates conformational changes of DNA-PKcs induced by KU70/80-DNA. The directions of movement of structural units are indicated with arrows. ⊗ represents the direction of movement that is downward perpendicular to plane of paper. (D-E) The kinase domain in the apo form (D) and the holo form (E) shown in similar orientations. The distance between FRBα4 and LBEα1, two α-helices on top of both sides of the groove, is shown for each kinase domain, indicating obviously distinct substrate entry grooves. The same conclusion can be obtained using molecule A (PDB: 5LUQ) for comparison (Supplementary information, Figure S8).

Figure 6

A working model for DNA-PK complex assembly and activation. When DNA DSB occurs, KU70/80 heterodimer recognizes the broken DNA ends and binds to the DNA with the preformed ring structure (left). DNA-PKcs is then recruited to the DSB end and binds to KU70/80-DNA (right). KU70/80-DNA stimulates the kinase activity of DNA-PKcs through an allosteric activation mechanism. DNA-PKcs and KU70/80 together bind to DNA and prevent sliding inward of DNA-PKcs along with DNA duplex, suggesting a mechanism by which the broken DNA end is protected from unnecessary processing.