February 2010

The three-dimensional structure of Death-Associated Protein Kinase (coloured in a rainbow from N- to C- terminal residues) when bound to calmodulin (green, calcium ions magenta).  This structure shows how calmodulin is able to activate DAPK kinase by binding to an autoregulatory domain (red). See de Diego et al. Sci. Signal. 3, ra6 (2010). Data were collected on beamlines BM14 and ID29 at the ESRF as well as at DESY.

Molecular Basis of the Death-Associated Protein Kinase–Calcium/Calmodulin Regulator Complex

The large and ubiquitous protein kinases family regulates the majority of cellular pathways and especially those involved in signal transduction.  One important subfamily is the calcium/calmodulin (CaM)–dependent protein serine-threonine kinases (CaMKs). An important structural feature of the CaMKs is an extra ‘autoregulatory domain’ (ARD) C-terminal to the catalytic domain. The CaMKs are activated when there is a flood of calcium ions inside the cell that are picked up by the CaM. It has been previously shown that different sites in the ARD are known to be important for the binding of CaM to CaMKs and their subsequent activation. The structure of death-associated protein kinase (DAPK) in complex with CaM shows how the ARD domain facilitates such an interaction. These results provide an important breakthrough in understanding the molecular basis of the more general CaM-CaMKs regulatory system. 

Data were collected on the beamlines BM14 and ID29 at the ESRF as well as at DESY.

January 2010

The structure of DNA-PKcs.  Proteins are coloured by chain.  This low resolution (dmin = 6.6 Å) structure provides the first description of the tertiary and quaternary structure of this important DNA binding protein.  See Sibanda et al. Nature, 463, 118-121 (2010). The data were collected on ID29.

The structure of DNA-PKcs provides the first detailed image of an important DNA repair protein

Breaks in cellular DNA that arise from reactive oxygen species or ionising radiation can lead to broken chromosomes that in turn may lead to cancer.  DNA dependent protein kinase (DNA-PK) senses the breaks in DNA and transmits this information, via phosphorylation, to other enzymes that prevent the cell cycle continuing.  The complex is very large and therefore obtaining crystals was challenging, over 2000 crystals were evaluated using the highly automated environment of the Structural Biology beamlines, and the best only diffracted X-rays to a resolution of 6.6 Å.  At this resolution only the rough fold of the the DNA-PK catalytic subunit (DNA-PKcs) can be seen. However, the crystal structure has provided important information on the potential mechanism of DNA break recognition.  Repeated helix-turn-helix motifs allow the protein to adopt a circular structure that may surround DNA and allow breaks to be repaired.

Data were collected on beamline ID29

December 2009

The structure of RNA polymerase II bound to transcription factor IIB.  RNA polymerase is coloured in a rainbow from its N- to C-terminus and transcription factor IIB is grey.  See Kostrewa et al. Nature, 462, 323-330 (2009).  The data were collected on beamline ID29.

The structure of eukaryotic RNA polymerase II in complex with transcription factor IIB provides a model for the transcription of the genetic code.

In order for proteins to be produced from the corresponding DNA it must first be transcribed into RNA.  This is performed in eukaryotes by RNA polymerase II (Pol II) that reads the DNA and creates a corresponding strand of RNA that is then used as a template to produce the protein.  Transcription factors play a vital role in initiating this process as they bind to both Pol II and promoter DNA forming a complex called the pre-initiation complex (PIC).  Researchers from Ludwig-Maximilians-Universität, Munich have solved the structure of S. cerevisiae Pol II bound to a transcription factor.  The structure of this complex provides the first model of  transcription initiation demonstrating which parts of the complex are responsible for  template DNA opening and set the scene for investigations into the regulation of cellular gene expression.

Data were collected on beamline ID29

November 2009

The structure of the myosin VI lever arm.  Calmodulin domains are coloured blue and red and the three helix bundle, that forms the lever arm extension, is coloured orange.  See Mukherjea et al. Mol. Cell, 35, 305-315 (2009). Data were collected on beamline ID23-1.

The structure of Myosin VI suggests dimerisation triggers an unfolding of a three-helix bundle in order to extend its step size.

Myosin VI is a class of myosin motor that moves toward the minus end of actin filaments and is crucial to transport within eukaryotic cells . It has a broad distribution of step sizes, around 30-36 nm, that is unusual compared to others myosins. A domain that is predicted to form an extended, stable alpha-helix (SAH) following the lever arm may be important in the mechanism. The region following the lever arm is alpha helical and forms a highly compacted domain, which in recent studies appeared to be a three-helix bundle. The region in between the three-helix bundle and the cargo-binding domain is a SAH that forms the bulk of the lever arm extension (LAE) necessary for the large step size of myosin VI.

In order to understand the mechanism that allows myosin VI to achieve its large step size  truncations were made at the C-terminus in order to define the location of the LAE and the minimal length structure that is capable of dimerising with a normal step size.

A truncated construct, including the region of 80 amino acids following the lever arm, was crystallized and the structure solved. The structure confirms that the region following the lever arm is a three-helix bundle.  Together with biochemical and fluorescence data the crystal structure indicates a new mechanism, where the three-helix bundle must unfold and form the extension of the myosin VI lever arm in order to achieve the larger step sizes.

Data were collected on beamline ID23-1

October 2009

Surface representation of the elongated tetramer depicted from the front (left) and rotated by about 90° (right). The basic-helical hairpin as RNA-binding motif is highlighted in red. The indicated distance shows the dimension of the continuous RNA-binding surface on both sides of the tetramer. See Müller, M., et al., RNA 15, (2009)

Formation of She2p tetramers is required for mRNA binding, mRNP assembly, and localization.

For transport, ASH1 mRNA in Saccharomyces cerevisiae is bound by the unusual RNA-binding protein She2p. Previous results indicated that She2p forms dimers required for RNA binding and transcript localization. Ultracentrifugation implied that She2p forms larger oligomeric complexes in solution and size-exclusion chromatography suggested that She2p forms tetramers at physiological concentrations (230nM). SAXS expeiments have demonstrated that the previously observed She2p dimers interact in a head-to-head conformation to form an elongated tetrameric complex. This suggests the generation of large continuous RNA-binding surfaces at both sides of the complex. Biochemical studies and immunostaining of cells confirmed that She2p tetramer formation is required for RNA binding, efficient mRNP assembly, and mRNA localization in vivo.

Part of SAXS data for this study were collected on ID14-3 at the ESRF.

September 2009

Secondary structure of 9-1-1 ring, coloured as follows: Rad9, green; Rad1, blue; and Hus1, magenta.  See Dore, A.S. et al., Mol. Cell 34, 735-745 (2009)

Crystal structure of the Rad9-Rad-1-Hus-1 DNA Damage Checkpoint Complex - Implications for Clamp loading and Regulation

Human Rad9, Rad1, and Hus1 form a heterotrimeric complex (9-1-1) that is loaded onto DNA at sites of DNA damage. DNA-loaded 9-1-1 activates the cell-cycle checkpoint kinase ATR, resulting in a signaling cascade that prevents cell-cycle progression. Evidence is accumulating that 9-1-1, like its distant relative PCNA, acts as a recruitment factor for translation DNA polymerases and a range of enzymes primarily involved in ‘‘long-patch’’ DNA base-excision repair. The crystal structure of the human Rad9-Rad1-Hus1 heterotrimeric complex (lacking the unstructured Rad9 C-terminal tail) at 2.9A° resolution, reveals a toroidal structure related to PCNA and other DNA clamps, but with marked asymmetry. The structure explains the formation of a unique heterotrimer and reveals significant differences between the subunits, suggesting a high degree of functional specialisation in their interactions with the clamp loader and other ligand proteins.

Data for this structure were collected on ID14-1 and ID29 at the ESRF.

July/August 2009

The overall complex structure of Toll-like receptor 4, myeloid differentiation factor 2 (ribbons) and lipopolysaccharide (spheres). See Beom, S.P., et al. Nature, 458, 1191-1195 (2009).

The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex

The lipopolysaccharide (LPS) of Gram negative bacteria is an inducer of the innate immune response. Toll-like receptor (TLR) 4 and myeloid differentiation factor 2 (MD-2) form a recognizes these structurally diverse LPS molecules. The TLR4–MD-2–LPS structure indicates a remarkable versatility of ligand recognition mechanisms employed by the TLR family, which is essential for defence against diverse microbial infection. The crystal structure of the TLR4–MD-2–LPS complex was determined at 3.1A ° resolution and the receptor multimer is composed of two copies of the TLR4–MD-2–LPS complex arranged in a symmetrical fashion. Comparison with published structure of the monomeric TLR4 and MD-2 complex shows that the overall folding of TLR4 and MD-2 is not disturbed by LPS binding or dimerisation.

Data for this structure were collected at the microfocus beamline ID23-2 at the ESRF and beamline 4A at Pohang Accelerator Laboratory

June 2009

The structure of yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex.  The complex is central to integrating transcription, processing, and mRNA nuclear export.  See Jani, D. et al. Mol. Cell, 33, 727-737 (2009).

The structural basis mRNA export from the nucleus.

The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores that is central to integrating transcription, processing, and mRNA nuclear export. Crystal structures for the Sac3:CID:Sus1:Cdc31 complex have been solved by researchers from the MRC laboratory of Molecular Biology (UK) and Heidelberg University (Germany). The CID region of Sac3 forms a continuous 12.5 nm alpha helix, which is encircled by two Sus1 chains and one Cdc31 chain. Data for these structures were collected on the ESRF beamlines ID23-1, ID29 and ID14-1.

May 2009

The structure of Olgopeptide binding protein A from Lactococcus lactis (ribbon) bound to a peptide substrate (spheres).  See Berntsson, R. P. et al. EMBO J (2009), doi:10.1038/emboj.2009.65

The structural basis of peptide selection by the transport receptor OppA

 Oligopeptide binding protein A (OppA) from Lactococcus lactis binds to a large range of peptides (of lengths varying from 4 to 35 residues), using no clear sequence dependence.  It achieves this by using a large cavity that allows the binding of large peptides without any constraints on the N or C termini of the ligand. A set of crystal structures has been reported in both the open and closed-liganded conformations. These structures demonstrate that the composition of the peptide, and not its actual sequence, is important for binding. OppA seems to prefer proline rich peptides containing at least one isolucine. This also correlates with the observed tendency of the organism to prefer proline rich proteins, such as caseins, as a source of amino acids.  Data for these structures were collected on the ESRF beamlines ID14-2 and ID29.

April  2009

Crystal structure of ZP3 ZP-N domain.  The structure is a novel Ig-like fold, with the strands A, B, E, D defining a standard Ig fold and the strands E’, F and G defining a β-sheet extension.  Two disulfide bridges between C46-C139 and C78-C98 stabilize the structure.  See Monne et al. Nature (2008) 456, 653-657

In mammals, the interaction between the egg extracellular matrix (Zona Pellucida) and the sperm is mediated by two components of the Zona Pellucida, ZP2 and ZP3, which act as sperm receptors. These proteins polymerise through a “Zona Pellucida domain” (ZP domain) whose conserved N-terminal part ZP-N constitutes a domain on its own.

Researchers from the Karolinska Institut in Hälsovägen (Sweden) have crystallized and solved the structure of the ZP3 ZP-N domain in three different crystal form.  The structure describes a novel Ig-like fold with the presence of a three β-strands platform (the EFG extension) containing an invariant Tyrosine residue essential for polymerization.

This is the first crystal structure of a protein essential for mammalian fertilization and it provides an important framework for understanding human pathologies caused by mutations in ZP domain-containing proteins. Data were collected at BM14 and ID23-1.

February / March 2009

The structure of the E.coli cell division protein FtsP.  The protein is structurally similar to multicopper oxidases but does not bind the copper cofactors, suggesting a structural role in  septal ring formation.  See Tarry et al. J. Mol. Biol. (2009) 386, 504-519

The Escherichia coli Cell Division Protein and Model Tat Substrate SufI (FtsP) Localizes to the Septal Ring and Has a Multicopper Oxidase-Like Structure

Around 20 proteins in E. coli are associated with the formation of the septal ring, a structure that forms in the midcell before cell division and constricts as the cell divides.  One of these proteins is SufI (FtsP), understanding its function will help to explain the mechanism of cell division in eubacteria.  Researchers from  the University of Oxford have solved the structure of FtsP and found it to be structurally similar to multicopper oxidases but lacking the metal cofactors.  Combined with biochemical data a role in stabilizing the septal ring has been proposed.  Data were collected at the European Synchrotron Radiation Facility (ESRF) on beamlines ID23-1 and ID29.

December 2008/January 2009

The X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. SeeNature (2008) doi:10.1038/nature07462

Towards a better understanding of the mechanism of ion permeation and gating during fast chemo-electrical transduction

Pentameric ligand-gated ion channels (pLGICs) are allosteric proteins regulating cellular excitability through the opening of an intrinsic transmembrane ion channel. Agonist binding to extracellular sites shifts a closed conformation into an open one, allowing ions to diffuse down their electrochemical gradient. Researchers from the Pasteur Institute and CNRS, Paris have recently solved the structure of a pentameric ligand-gated ion channel for the first time in the open conformation. Structural comparison with a homologue protein observed in a presumed closed conformation suggests a possible mechanism for ion permeation. Data were collected at the European Synchrotron Radiation Facility (ESRF) on the microfocus beamline ID23-2

October/November 2008

The structure of the E. coli mechanoselective channel MscS solved at the ESRF. SeeScience (2008) 321, 1179-1183

Scientists have worked out a key mechanism that protects bacteria against stress in a major discovery that could lead to new ways of killing superbugs C. difficile and MRSA.

Researchers from the Universities of St Andrews and Aberdeen have discovered the mechanism of a pressure-release valve - which helps safeguard bacteria. The findings of the two teams led by Professor James Naismith (St Andrews) and Professor Ian Booth (Aberdeen), could pave the way for new chemicals to combat potentially deadly bugs.  Possible applications could be as basic as cleansing hospital equipment and wards or helping to make food safer. The structure was solved from data collected at the European Light Source (ESRF) at beamline ID14-2.