title: The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR).
abstract: In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopy we localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.
authors.affiliation: Bloomsbury Centre for Structural Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Harris
authors.forename: Diego
authors.initials: D
authors.lastname: Esposito
authors.forename: Andrew
authors.initials: A
authors.lastname: Sankar
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Maman
authors.forename: John A
authors.initials: JA
authors.lastname: Hinks
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Pearl
authors.forename: Paul C
authors.initials: PC
authors.lastname: Driscoll
mesh_terms: D000595:Amino Acid Sequence; D034961:Antigens, Nuclear / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D000072200:Ku Autoantigen; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D039502:Nuclear Cap-Binding Protein Complex / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012996:Solutions
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D039502:Nuclear Cap-Binding Protein Complex; D010750:Phosphoproteins; D012996:Solutions; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen