Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats

Abstract

Broken chromosomes arising from DNA double-strand breaks result from endogenous events such as the production of reactive oxygen species during cellular metabolism, as well as from exogenous sources such as ionizing radiation. Left unrepaired or incorrectly repaired they can lead to genomic changes that may result in cell death or cancer. DNA-dependent protein kinase (DNA-PK), a holoenzyme that comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer Ku70/Ku80, has a major role in non-homologous end joining-the main pathway in mammals used to repair double-strand breaks. DNA-PKcs is a serine/threonine protein kinase comprising a single polypeptide chain of 4,128 amino acids and belonging to the phosphatidylinositol-3-OH kinase (PI(3)K)-related protein family. DNA-PKcs is involved in the sensing and transmission of DNA damage signals to proteins such as p53, setting off events that lead to cell cycle arrest. It phosphorylates a wide range of substrates in vitro, including Ku70/Ku80, which is translocated along DNA. Here we present the crystal structure of human DNA-PKcs at 6.6 A resolution, in which the overall fold is clearly visible, to our knowledge, for the first time. The many alpha-helical HEAT repeats (helix-turn-helix motifs) facilitate bending and allow the polypeptide chain to fold into a hollow circular structure. The carboxy-terminal kinase domain is located on top of this structure, and a small HEAT repeat domain that probably binds DNA is inside. The structure provides a flexible cradle to promote DNA double-strand-break repair.

Figure 1. Crystal structure of DNA-PKcs at 6.6Å resolution

a) Molecular surface of the two DNA-PKcs molecules present in the asymmetric unit of the crystal that are related by the two-fold non-crystallographic symmetry. The colour-coding of the molecule is as follows: green – the ring structure; light green – the forehead that is part of the ring structure; cyan – the putative DNA-binding domain; magenta – the larger C-terminal region that carries the FAT and FATC domains; yellow – the kinase domain. Stereo diagrams of b) a representative area of SigmaA weighted 2F_o-F_c electron density map from final refinement in the kinase domain region (ribbon representation in yellow colour); c) experimental F_o electron density map calculated with phases obtained by MAD method in the area of the N-terminal HEAT repeat ring, the final DNA-PKcs model shown in green. Both electron density maps shown are contoured at 1.0 sigma level.

Figure 2. Overall view of the DNA-PKcs structure

Molecular surface of the DNA-PKcs: a) front of the molecule. The colour-coding of the different parts of the molecule is as shown in figure 1a. b) Side view. c) Ribbon representation of the Cα positions of figure 2a, indicating the overall size and the sites that are potentially flexible at 135° and 225°. Stereo diagrams of d) experimental F_o electron density map in the HEAT repeat area of the ring structure with the final DNA-PKcs model shown in green; e) HEAT repeats of phosphatase 2A PR65/A subunit (residues 1 to 325; PDB accession code 1b3u) superposed onto the electron density of DNA-PKcs structure. The N- and C-termini of phosphatase 2A PR65/A subunit are shown. Both electron density maps are contoured at 1.0 sigma level.

Figure 3. Location of the DNA-PKcs kinase domain

a) The overall structure of DNA-PKcs showing the location of the kinase catalytic domain (yellow). The two helices of the N-lobe are shown in brown and were not built into the final structure of DNA-PKcs due to unclear electron density in this area. The catalytic domain of DNA-PKcs was built based on the crystal structure of PI3Kγ kinase (PDB accession code: 1e8x). b) A close up view of the DNA-PKcs kinase catalytic domain. Two helices of PI3Kγ kinase N-lobe could occupy the positions of helices 1 and 2 of DNA-PKcs shown in magenta.