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

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

Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: Homology of the AhpD-like structural family

<p>Abstract</p> <p>Background</p> <p>Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein.</p> <p>Results</p> <p>The crystal structure of a putative AhpD from <it>Pseudomonas aeruginosa </it>has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from <it>Mycobacterium tuberculosis </it>despite a low overall sequence identity of 9%. A conserved two α-helical motif responsible for function is present in both. However, in the <it>P. aeruginosa </it>protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in <it>M. tuberculosis </it>AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of <it>P. aeruginosa </it>have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from <it>P. aeruginosa </it>exhibits a weak ability to reduce H₂O₂as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents.</p> <p>Conclusion</p> <p>Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.</p

Achieving high signal-to-noise in cell regulatory systems: Spatial organization of multiprotein transmembrane assemblies of FGFR and MET receptors.

How is information communicated both within and between cells of living systems with high signal to noise? We discuss transmembrane signaling models involving two receptor tyrosine kinases: the fibroblast growth factor receptor (FGFR) and the MET receptor. We suggest that simple dimerization models might occur opportunistically giving rise to noise but cooperative clustering of the receptor tyrosine kinases observed in these systems is likely to be important for signal transduction. We propose that this may be a more general prerequisite for high signal to noise in transmembrane receptor signaling.D.B.A is the recipient of a C. J. Martin Research Fellowship from the National Health and Medical Research Council of Australia (APP1072476). TLB and MB receive funding from the Gates Foundation, and T.L.B. and D.Y.C. from The Wellcome Trust (093167) for facilities and support. D.Y.C. is also supported by the Crystallographic X-ray Facility, Department of Biochemistry, University of Cambridge. We thank Ermanno Gherardi for many contributions to the experiments and to our thinking on the Met receptor structure and activation over the years.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.pbiomolbio.2015.04.00

CryoEM structures of human CMG-ATPγS-DNA and CMG-AND-1 complexes.

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one β-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins

Structural biology of DNA repair: spatial organisation of the multicomponent complexes of nonhomologous end joining.

Nonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV) is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods—X-ray crystallography, electron microscopy and small angle X-ray scattering—can give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK) and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higher-order complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere

Achieving selectivity in space and time with DNA double-strand-break response and repair: molecular stages and scaffolds come with strings attached

When double-strand breaks (DSBs) in DNA remain unrepaired, catastrophic loss of genes occurs, leading to translocations, mutations and carcinogenesis. If a sister chromatid is not available at the DNA DSB, nonhomologous end joining (NHEJ) is used to join broken ends. The NHEJ pathway comprises synapsis, end processing and ligation. Here, we ask how DSBs in DNA are repaired efficiently. We suggest that colocation of proteins is achieved over time by the following components: stages, where the main actors are assembled, scaffolds that are erected quickly around broken parts to give access, and strings that tether proteins together. In NHEJ, a $\textit{stage}$ is provided by the Ku heterodimer interacting with DSBs and several other proteins including DNA-PKcs, APLF, BRCA1 and PAXX. A further $\textit{stage}$, DNA-PKcs, links the kinase with DNA, Ku, PARP1, BRCA1 and Artemis. A temporary $\textit{scaffold}$ facilitates repair and is constructed from XRCC4/XLF filaments that bridge Ku bound at DSB ends. LigIV bound to XRCC4 C-termini likely terminates the scaffold, bringing LigIV close to the DNA broken ends. A $\textit{string}$, provided by the Artemis C-terminal region, is intrinsically disordered but includes short linear ‘‘epitopes’’ that recognise DNA-PKcs, LigIV and PTIP, so keeping these components nearby. We show that these stages, scaffolds and strings facilitate colocation and efficient DSB repair. Understanding these processes provides insight into the biology of DNA repair and possible therapeutic intervention in cancer and other diseases.Wellcome Trust (Programme Grant ID: 093167/Z/ 10/Z), National Health and Medical Research Council of Australia (C. J. Martin Research Fellowship, Grant ID: APP1072476), Gates Cambridge ScholarshipThis is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s11224-016-0841-

Structural insights into the role of domain flexibility in human DNA ligase IV.

Knowledge of the architecture of DNA ligase IV (LigIV) and interactions with XRCC4 and XLF-Cernunnos is necessary for understanding its role in the ligation of double-strand breaks during nonhomologous end joining. Here we report the structure of a subdomain of the nucleotidyltrasferase domain of human LigIV and provide insights into the residues associated with LIG4 syndrome. We use this structural information together with the known structures of the BRCT/XRCC4 complex and those of LigIV orthologs to interpret small-angle X-ray scattering of LigIV in complex with XRCC4 and size exclusion chromatography of LigIV, XRCC4, and XLF-Cernunnos. Our results suggest that the flexibility of the catalytic region is limited in a manner that affects the formation of the LigIV/XRCC4/XLF-Cernunnos complex

A new look at sodium channel β subunits.

Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits