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Resolution Crystal Structure (resolution + crystal_structure)
Selected AbstractsThe structure of the human allo-ligand HLA-B*3501 in complex with a cytochrome p450 peptide: Steric hindrance influences TCR allo-recognitionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2006Christopher Abstract Virus-specific T cell populations have been implicated in allo-recognition. The subdominant T cell receptor JL12 recognizes both HLA-B*0801 presenting the Epstein,Barr virus-derived peptide FLRGRAYGL and also HLA-B*3501 presenting the cytochrome p450 self peptide KPIVVLHGY. This cross-reactivity could promote the rejection of HLA-B*3501-positive cells in Epstein,Barr virus-exposed HLA-B*0801 recipients. LC13, the dominant TCR against the HLA-B*0801:FLRGRAYGL complex, fails to recognize HLA-B*3501:KPIVVLHGY. We report the 1.75-Angstrom resolution crystal structure of the human allo-ligand HLA-B*3501:KPIVVLHGY. Similarities between this structure and that of HLA-B*0801:FLRGRAYGL may facilitate cross-recognition by JL12. Moreover, the elevated peptide position in HLA-B*3501:KPIVVLHGY would provide steric hindrance to LC13, preventing it from interacting in the manner in which it interacts with HLA-B*0801:FLRGRAYGL. These findings are relevant to understanding the basis of T cell cross-reactivity in allo-recognition, optimal transplant donor-recipient matching and developing specific molecular inhibitors of allo-recognition. [source] Rapid purification of active ,-secretase, an intramembrane protease implicated in Alzheimer's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Matthias Cacquevel Abstract ,-Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-, precursor protein generates the amyloid-, protein (A,) of Alzheimer's disease, partially inhibiting ,-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of ,-secretase, but at low resolution (15 Å). The limited amount of purified ,-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four ,-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active ,-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme. [source] Flexibility of the Cu,Zn superoxide dismutase structure investigated at 0.57,GPaACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010Isabella Ascone The 2,Å resolution crystal structure of bovine erythrocyte Cu,Zn superoxide dismutase (CuZnSOD) has been determined by X-ray diffraction at high pressure (0.57,GPa) and room temperature. At 0.57,GPa the secondary, tertiary and quaternary structures are similar to other previously determined bovine erythrocyte CuZnSOD structures. Nevertheless, pressure has a localized impact on the atomic coordinates of C, atoms and on side chains. The compression of the crystal and of the protein backbone is anisotropic. This anisotropy is discussed, taking into account intermolecular contacts and protein conformation. Pressure perturbation highlights the more flexible zones in the protein such as the electrostatic loop. At 0.57,GPa, a global shift of the dimetallic sites in both subunits and changes in the oxidation state of Cu were observed. The flexibility of the electrostatic loop may be useful for the interaction of different metal carriers in the copper-uptake process, whereas the flexibility of the metal sites involved in the activity of the protein could contribute to explaining the ubiquitous character of CuZnSODs, which are found in organisms living in very different conditions, including the deep-sea environment. This work illustrates the potential of combining X-ray crystallography with high pressure to promote and stabilize higher energy conformational substates. [source] Structure of the putative thioesterase protein TTHA1846 from Thermus thermophilus HB8 complexed with coenzyme A and a zinc ionACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Toshiaki Hosaka TTHA1846 is a conserved hypothetical protein from Thermus thermophilus HB8 with a molecular mass of 15.1,kDa that belongs to the thioesterase superfamily (Pfam 03061). Here, the 1.9,Å resolution crystal structure of TTHA1846 from T. thermophilus is reported. The crystal structure is a dimer of dimers. Each subunit adopts the so-called hot-dog fold composed of five antiparallel ,-strands flanked on one side by a rather long ,-helix and shares structural similarity to a number of thioesterases. Unexpectedly, TTHA1846 binds one metal ion and one ligand per subunit. The ligand density was modelled as coenzyme A (CoA). Its structure was confirmed by MALDI,TOF mass spectrometry and electron-density mapping. X-ray absorption fine-structure (XAFS) measurement of the crystal unambiguously characterized the metal ion as zinc. The zinc ion is tetrahedrally coordinated by the side chains of Asp18, His22 and Glu50 and the CoA thiol group. This is the first structural report of the interaction of CoA with a zinc ion. From structural and database analyses, it was speculated that the zinc ion may play an inhibitory role in the enzymatic activity. [source] Structure of ristocetin A in complex with a bacterial cell-wall mimeticACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Virginie Nahoum Antimicrobial drug resistance is a serious public health problem and the development of new antibiotics has become an important priority. Ristocetin A is a class III glycopeptide antibiotic that is used in the diagnosis of von Willebrand disease and which has served as a lead compound for the development of new antimicrobial therapeutics. The 1.0,Å resolution crystal structure of the complex between ristocetin A and a bacterial cell-wall peptide has been determined. As is observed for most other glycopeptide antibiotics, it is shown that ristocetin A forms a back-to-back dimer containing concave binding pockets that recognize the cell-wall peptide. A comparison of the structure of ristocetin A with those of class I glycopeptide antibiotics such as vancomycin and balhimycin identifies differences in the details of dimerization and ligand binding. The structure of the ligand-binding site reveals a likely explanation for ristocetin A's unique anticooperativity between dimerization and ligand binding. [source] 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cellsACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Kausik Chattopadhyay Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique `strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8,Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli -expressed proteins. The S2 cell-expressed murine GITRL adopts an identical `strand-exchanged' dimeric structure to that observed for the E. coli -expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL. [source] Conformational flexibility in the flap domains of ligand-free HIV proteaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2007Holly Heaslet The crystal structures of wild-type HIV protease (HIV PR) in the absence of substrate or inhibitor in two related crystal forms at 1.4 and 2.15,Å resolution are reported. In one crystal form HIV PR adopts an `open' conformation with a 7.7,Å separation between the tips of the flaps in the homodimer. In the other crystal form the tips of the flaps are `curled' towards the 80s loop, forming contacts across the local twofold axis. The 2.3,Å resolution crystal structure of a sixfold mutant of HIV PR in the absence of substrate or inhibitor is also reported. The mutant HIV PR, which evolved in response to treatment with the potent inhibitor TL-3, contains six point mutations relative to the wild-type enzyme (L24I, M46I, F53L, L63P, V77I, V82A). In this structure the flaps also adopt a `curled' conformation, but are separated and not in contact. Comparison of the apo structures to those with TL-3 bound demonstrates the extent of conformational change induced by inhibitor binding, which includes reorganization of the packing between twofold-related flaps. Further comparison with six other apo HIV PR structures reveals that the `open' and `curled' conformations define two distinct families in HIV PR. These conformational states include hinge motion of residues at either end of the flaps, opening and closing the entire ,-loop, and translational motion of the flap normal to the dimer twofold axis and relative to the 80s loop. The alternate conformations also entail changes in the ,-turn at the tip of the flap. These observations provide insight into the plasticity of the flap domains, the nature of their motions and their critical role in binding substrates and inhibitors. [source] Near-atomic resolution crystal structure of an A-DNA decamer d(CCCGATCGGG): cobalt hexammine interaction with A-DNAACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2003Boopathy Ramakrishnan The structure of the DNA decamer d(CCCGATCGGG) has been determined at 1.25,Å resolution. The decamer crystallized in the tetragonal space group P43212, with unit-cell parameters a = b = 44.3, c = 24.8,Å and one strand in the asymmetric unit. The structure was solved by the molecular-replacement method and refined to Rwork and Rfree values of 16.3 and 18.5%, respectively, for 5969 reflections. The decamer forms the A-form DNA duplex, with the abutting crystal packing typical of A-DNA. The crystal packing interactions seem to distort the local conformation: A5 adopts the trans/trans conformation for the torsion angles , and , instead of the usual gauche,/gauche+ conformations, yielding G*(G·C) base triplets. The highly hydrated [Co(NH3)6]3+ ion adopts a novel binding mode to the DNA duplex, binding directly to phosphate groups and connecting to N7 and O6 atoms of guanines by water bridges. Analysis of thermal parameters (B factors) shows that the nucleotides involved in abutting crystal packing are thermally more stable than other nucleotides in the duplex. [source] Atomic resolution crystal structure of squid ganglion DFPaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-1 2002Juergen Koepke Diisopropylfluorophosphatases (DFP-ases) are capable of detoxifying chemical warfare agents like diisopropylfluorophosphate (DFP) by hydrolysis. The protein reported here was recombinantely expressed in E. coli. The X-ray cystal structure of this enzyme has been refined to a resolution of 0.85 Å and a crystallographic R value of 9.4%. Reversible flash-cooling improved both, mosaicity and resolution of the crystals considerably. The overall structure of this protein represents a six-bladed ,-propeller with two calcium ions bound in a central water filled tunnel. 496 water, 2 glycerol, 2 MES-buffer molecules, and 18 PEG fragments of different lengths could be refined in the solvent region. The 208 most reliable residues, without disorder or reduced occupancy in their side-chains, were finally refined without restraints. A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (esds) by matrix inversion. The herewith calculated bond lenghts and bond-esds were used to obtain averaged bond lengths, which have been compared to the restraints used in preceding refinement cycles. [source] A conserved mechanism of autoinhibition for the AMPK kinase domain: ATP-binding site and catalytic loop refolding as a means of regulationACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Dene R. Littler The AMP-activated protein kinase (AMPK) is a highly conserved trimeric protein complex that is responsible for energy homeostasis in eukaryotic cells. Here, a 1.9,Å resolution crystal structure of the isolated kinase domain from the ,2 subunit of human AMPK, the first from a multicellular organism, is presented. This human form adopts a catalytically inactive state with distorted ATP-binding and substrate-binding sites. The ATP site is affected by changes in the base of the activation loop, which has moved into an inhibited DFG-out conformation. The substrate-binding site is disturbed by changes within the AMPK,2 catalytic loop that further distort the enzyme from a catalytically active form. Similar structural rearrangements have been observed in a yeast AMPK homologue in response to the binding of its auto-inhibitory domain; restructuring of the kinase catalytic loop is therefore a conserved feature of the AMPK protein family and is likely to represent an inhibitory mechanism that is utilized during function. [source] High-resolution structure of the antibiotic resistance protein NimA from Deinococcus radioduransACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2008Hanna-Kirsti S. Leiros Many anaerobic human pathogenic bacteria are treated using 5-nitroimidazole-based (5-Ni) antibiotics, a class of inactive prodrugs that contain a nitro group. The nitro group must be activated in an anaerobic one-electron reduction and is therefore dependent on the redox system in the target cells. Antibiotic resistance towards 5-Ni drugs is found to be related to the nim genes (nimA, nimB, nimC, nimD, nimE and nimF), which are proposed to encode a reductase that is responsible for converting the nitro group of the antibiotic into a nonbactericidal amine. A mechanism for the Nim enzyme has been proposed in which two-electron reduction of the nitro group leads to the generation of nontoxic derivatives and confers resistance against these antibiotics. The cofactor was found to be important in the mechanism and was found to be covalently linked to the reactive His71. In this paper, the 1.2,Å atomic resolution crystal structure of the 5-nitroimidazole antibiotic resistance protein NimA from Deinococcus radiodurans (DrNimA) is presented. A planar cofactor is clearly visible and well defined in the electron-density map adjacent to His71, the identification of the cofactor and its properties are discussed. [source] Crystal structures of the Arabidopsis thaliana proliferating cell nuclear antigen 1 and 2 proteins complexed with the human p21 C-terminal segmentPROTEIN SCIENCE, Issue 5 2009Wojciech Strzalka Abstract The proliferating cell nuclear antigen (PCNA) is well recognized as one of the essential cellular components of the DNA replication machinery in all eukaryotic organisms. Despite their prominent importance, very little biochemical and structural information about plant PCNAs is available, in comparison with that obtained from other eukaryotic organisms. We have determined the atomic resolution crystal structures of the two distinct Arabidopsis thaliana PCNAs (AtPCNA), both complexed with the C-terminal segment of human p21. Both AtPCNAs form homotrimeric ring structures, which are essentially identical to each other, including the major contacts with the p21 peptide. The structure of the amino-terminal half of the p21 peptide, containing the typical PIP box sequence, is remarkably similar to those observed in the previously reported crystal structures of the human and archaeal PCNA-PIP box complexes. Meanwhile, the carboxy-terminal halves of the p21 peptide in the plant PCNA complexes are bound to the protein in a unique manner, most probably because of crystal packing effects. A surface plasmon resonance analysis revealed high affinity between each AtPCNA and the C-terminal fragment of human p21. This result strongly suggests that the interaction is functionally significant, although no plant homologs of p21 have been identified yet. We also discovered that AtPCNA1 and AtPCNA2 form heterotrimers, implying that hetero-PCNA rings may play critical roles in cellular signal transduction, particularly in DNA repair. [source] | ||||||||||