Bancinyane L. Sibanda

Crystal structure of an Xrcc4-DNA ligase IV complex.

A complex of two proteins, Xrcc4 and DNA ligase IV, plays a fundamental role in DNA non-homologous end joining (NHEJ), a cellular function required for double-strand break repair and V(D)J recombination. Here we report the crystal structure of human Xrcc4 bound to a polypeptide that corresponds to the DNA ligase IV sequence linking its two BRCA1 C-terminal (BRCT) domains. In the complex, a single ligase chain binds asymmetrically to an Xrcc4 dimer. The helical tails of Xrcc4 undergo a substantial conformational change relative to the uncomplexed protein, forming a coiled coil that unwinds upon ligase binding, leading to a flat interaction surface. A buried network of charged hydrogen bonds surrounded by extensive hydrophobic contacts explains the observed tightness of the interaction. The strong conservation of residues at the interface between the two proteins provides evidence that the observed mode of interaction has been maintained in NHEJ throughout evolution.

Amino and carboxy-terminal regions in globular proteins

The structural, dynamic and functional aspects of amino and carboxy-terminal regions in proteins of known structure have been analysed. Terminal regions are usually located on the surface of the protein, accessible to solvent, and are often flexible. There is a significant preference for terminal regions in single domain proteins, and within individual domains of larger proteins, to be in close proximity. This partially reflects the compact globular nature of proteins, but the preference for spatial proximity is stronger in native proteins than in randomly generated structures. In addition in multi-domain and multi-subunit proteins we find that the terminal regions are commonly involved in the interface between domains and subunits. In the 18 multi-domain structures analysed, 19 terminal regions provide a link between domains. Subunit links are also frequently observed. In contrast, the distribution of active site residues along the sequence, indicates that the terminal regions are less frequently involved in activity. These data suggest that in many globular proteins the terminal regions fulfil a structural role, stabilizing the tertiary or quaternary structure to provide a framework for the active site.

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 Å resolution, in which the overall fold is clearly visible, to our knowledge, for the first time. The many α-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.

Structural biology and bioinformatics in drug design: opportunities and challenges for target identification and lead discovery

Impressive progress in genome sequencing, protein expression and high-throughput crystallography and NMR has radically transformed the opportunities to use protein three-dimensional structures to accelerate drug discovery, but the quantity and complexity of the data have ensured a central place for informatics. Structural biology and bioinformatics have assisted in lead optimization and target identification where they have well established roles; they can now contribute to lead discovery, exploiting high-throughput methods of structure determination that provide powerful approaches to screening of fragment binding.

Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ

The recently characterised 299‐residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non‐homologous end joining (NHEJ) and interacts with the XRCC4–DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1–233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled‐coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C‐terminal helical structure wedged between the coiled‐coil and head domain. The existence of a dimer as the major species is confirmed by size‐exclusion chromatography, analytical ultracentrifugation, small‐angle X‐ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino‐acid conservation and mutations in immunodeficient patients. Our data are most consistent with head‐to‐head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.

[5] Conformation of β hairpins in protein structures: Classification and diversity in homologous structures

Publisher Summary This chapter describes a systematic classification of β hairpins that is indicative of both the length of the polypeptide and the hydrogen bonding between the two antiparallel strands. The classification provides a useful tool to aid “modeling by homology” of protein structure. In this approach, the sequence of the “unknown” is modeled onto the three-dimensional coordinates provided by one or more homologous proteins. Comparisons of three-dimensional structures of homologous proteins demonstrate that the cores of proteins are highly conserved, although there are differences in relative positions and orientations of α helices and β strands. Insertions, deletions, and radical changes in conformation due to replacement are largely confined to the loop regions. These loops are, therefore, the most difficult to model and both knowledge-based and theoretical molecular mechanics/dynamics methods are being developed. The knowledge-based approach involves specifying the end-point geometry of the loop, its length, and sequence, and then searching in a database of known structures for loops, which fulfill these criteria. To explore the way changes in sequence are accommodated in β hairpins in known structures, a comparison has been drawn among the conformations of homologous β hairpins in several families. The data illustrates the way the hairpins move between two classes as residues are added or deleted and can adopt the common loop family structures if the sequence is appropriate.

DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair

Activating DNA repair DNA double-strand breaks must be repaired efficiently to avoid cell death or cancer. The break ends can either be directly ligated by nonhomologous end joining (NHEJ) or more accurately repaired by homologous recombination that uses information from the sister chromatid. Sibanda et al. present a high-resolution x-ray structure of a key component of the DNA repair machinery, the DNA-dependent kinase catalytic subunit (DNA-PKcs), bound to a C-terminal peptide of Ku80. The structure suggests that Ku80 presents the DNA ends for repair to a DNA-PKcs dimer and that activity is modulated by interactions between the monomers. Binding of either Ku80 or BRCA1, which may compete for the same binding site on DNA-PKcs, could provide a switch between NHEJ and homologous recombination. Science, this issue p. 520 A crystal structure shows how components of the nonhomologous end-joining machinery modulate kinase activation in DNA repair. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128–amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.

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.

The Aspartic Proteinases

Research into the aspartic proteinases has had a rich and diverse history. It began with studies of the digestive juices of man and nepenthes using somewhat primitive technologies. It had a renaissance of biochemical characterisation, followed by a classical period when sequences were defined and catalytic activity identified. It is now very much under the influence of the structural school. Thus, in many ways its history parallels that of painting. And like painting it is now thoroughly commercialised, a mixed blessing which nevertheless keeps many of its practioners well above the breadline.

Spatial and temporal organization of multi-protein assemblies: achieving sensitive control in information-rich cell-regulatory systems.

The regulation of cellular processes in living organisms requires signalling systems that have a high signal-to-noise ratio. This is usually achieved by transient, multi-protein complexes that assemble cooperatively. Even in the crowded environment of the cell, such assemblies are unlikely to form by chance, thereby providing a sensitive regulation of cellular processes. Furthermore, selectivity and sensitivity may be achieved by the requirement for concerted folding and binding of previously unfolded components. We illustrate these features by focusing on two essential signalling pathways of eukaryotic cells: first, the monitoring and repair of DNA damage by non-homologous end joining, and second, the mitotic spindle assembly checkpoint, which detects and corrects defective attachments of chromosomes to the kinetochore. We show that multi-protein assemblies moderate the full range of functional complexity and diversity in the two signalling systems. Deciphering the nature of the interactions is central to understanding the mechanisms that control the flow of information in cell signalling and regulation.