Chemistry of 1,3-Dioxepins. XIV.1 Structural Studies of 4,7-Dihydro-(4,7-dihydro-1,3-dioxepin-2-yl)-1,3-dioxepin and Their Metal Complexes

The coordination properties and conformational behaviour of 4,7- dihydro-(4,7-dihydro-1,3-dioxepin-2-yl)-1,3-dioxepin 1, bis-(1,3-dioxepin), or C10H14O4 have been investigated. The crystal and molecular structures of bis-(1,3-dioxepin) (1), bis-(1,3-dioxepin) lithiumperchlorate (2:1) (2) and bis-(1,3-dioxepin) sodium-perchlorate (1:1) (3) complexes, as well as bis-(1,3-dioxepin) hexsaaquamagnesiumperchlorate (3:1) chlatrate (4), have been determined by X-ray diffraction. The molecule of bis-(1,3-dioxepin) is a bidentate ligand in mononuclear lithium complex 2, in contrast to its bridging mode in 2D polymeric sodium complex 3. The coordination environment around lithium in 2 is distorted octahedral with C2 symmetry, while around sodium in 3 it is quasi-pentagonal bipyramidal. Out of four possible conformations of bis-(1,3-dioxepin) molecule, twist-boattwist- boat centrosymmetric (tb-tb-c), twist-boat-twist-boat non-centrosymmetric (tb-tb), twist-boat-chair (tb-ch) and chair-chair (ch-ch), three have been found in the investigated crystal structures: ch-ch in 1, tb-tb in 2 and tb-tb-c in 3 and 4

Methionine Adenosyltransferase Purified from Rat Liver

Methionine adenosyltransferase (MAT III), also known as S-adeno-sylmethionine synthetase, was purified from rat liver and crystallized. X-ray diffraction data were collected using a microfocused synchrotron radiation. The crystallization conditions were extensively optimized but final crystal size was never larger than 303 pm3. Due to their small size crystals had no detectable diffraction on either rotating anode source or the Deresbury SRS beamline 9.6 (GB). Finally, four data sets were collected on Grenoble ESRF (France) undulator microfocus beamline ID13 to resolution of 3.2-3.6 Å. Crystals belong to the cubic space group F432 with cell dimension a = 246 Å. Attempts are under way to solve the structure by molecular replacement, using recombinant MAT I rat liver structure as a search model

Mass Spectrometry Reveals Protein Kinase CK2 High-Order Oligomerization via the Circular and Linear Assembly.

CK2 is an intrinsically active protein kinase that is crucial for cellular viability. However, conventional kinase regulatory mechanisms do not apply to CK2, and its mode of regulation remains elusive. Interestingly, CK2 is known to undergo reversible ionic-strength-dependent oligomerization. Furthermore, a regulatory mechanism based on autoinhibitory oligomerization has been postulated on the basis of the observation of circular trimeric oligomers and linear CK2 assemblies in various crystal structures. Here, we employ native mass spectrometry to monitor the assembly of oligomeric CK2 species in an ionic-strength-dependent manner. A subsequent combination of ion mobility spectrometry-mass spectrometry and hydrogen-deuterium exchange mass spectrometry techniques was used to analyze the conformation of CK2 oligomers. Our findings support ionic-strength-dependent CK2 oligomerization, demonstrate the transient nature of the α/β interaction, and show that CK2 oligomerization proceeds via both the circular and linear assembly.This research was supported by the Wellcome Trust Strategic Award (090340/Z/09/Z), the Agency for Science Technology and Research (A*STAR) Singapore (Ph.D. sponsorship, W.G.S.), and the Croucher Foundation and the Cambridge Overseas Trust (Croucher Cambridge International Scholarship, D.S.-H.C).This is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/acschembio.6b0006

Crystallographic Study of Mutant Lysl20Leu Xenopus laevis Cu,Zn Superoxide Dismutase

Theoretical calculations and experimental measurements on the Xenopus laevis Cu,Zn superoxide dismutase (XSODB) wild-type protein and on some of its engineered mutants showed that the electrostatic arrangement around the active site channel plays a fundamental role in determining the catalytic properties of the en-zyme. Lysl20, which lies on the lip of the active site channel, about 11 Å from the catalytic copper ion, influences the enzyme electrostatic environment and binding selectivity. Neutralization of this residue has the effect of decreasing the activity of the enzyme versus the negatively charged substrate. In order to get precise information about the mutated residue and its effects on the structure of the engineered protein, the crystal structure of single site Lysl20Leu mutant XSODB was determined at 2.0 Å resolution, and refined to an R-factor value of 0.181. The structure of Lysl20Leu mutant XSODB is little affected by the amino-acid substitution, suggesting that the main effect of the mutation is perturbation of the electrostatic properties of the SOD catalytic center

Conformational Behaviour of 11-O-Methylazithromycin in the Solid and Solution State

Conformational behaviour of azithromycin 11-OMe derivative 2 (Scheme 1) has been studied in the solid and solution state. In the CDCl3 and DMSO solution, 2 mainly adopts the »folded-in« conformation. 11-OMe group is oriented toward the centre of aglycone ring. The crystal structure of DMSO solvate of 2 has been solved by the molecular replacement method using the solution state conformation as the search model. Conformation of 2 in the solid and solution state is very similar. Molecules of 2 are held together in the crystal by van der Waals interactions, forming a solvent channel along the b axis. The DMSO molecule is found to be disordered and bound to cladinose moiety of 2 by H-bond O4"-H...01s

Structure of a single-chain Fv bound to the 17 N-terminal residues of huntingtin provides insights into pathogenic amyloid formation and suppression.

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3-11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12-17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv-peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.E.D.G. and C.M.D. are grateful for support by the Medical Research Council (G1002272). We also thank the Hereditary Disease Foundation (A.M.). D.Y.C. is supported by the Crystallographic X-ray Facility at the Department of Biochemistry, University of Cambridge. We would like to acknowledge Dr. Katherine Stott at the Biophysics Facility at the Department of Biochemistry, University of Cambridge, for her help with the ultracentrifugation experiments and Prof. Weiss and Dr. Desplancq at the Ecole Supérieure de Biotechnologie de Strasbourg for the kind gift of the gankyrin-specific scFv, scFvR19 as a control for our in vitro aggregation experiments.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S002228361500217X#

Research data supporting "Mass spectrometry reveals protein kinase CK2 high–order oligomerization via the circular trimeric assembly"

Raw or processed data derived from native mass spectrometry, ion–mobility spectrometry–mass spectrometry and HDX–MS experiments organized into 4 folders. Folder name: Excess CK2alpha • Raw data of native mass spectrometry experiments with excess CK2α • E.g. CK2alpha-0.40AA.raw • AA refers to ammonium acetate • Numbers refer to concentration of ammonium acetate Folder name: Excess CK2beta • Raw data of native mass spectrometry experiments with excess CK2β • E.g. CK2beta-0.20AA.raw • AA refers to ammonium acetate • Numbers refer to concentration of ammonium acetate Folder name: HDX • Processed HDX data using CK2α as search sequence Folder name: Ion mobility • Raw data of ion mobility experiments • E.g. AH-H7.raw • First identifier (e.g. AH) refers to protein • Second identifier (e.g. H7) refers to wave height • AH = alcohol dehydrogenase • AVD = avidin • CCA = concanavalin A • PK = pyruvate kinaseFolder name: Excess CK2alpha • Raw data of native mass spectrometry experiments with excess CK2α • E.g. CK2alpha-0.40AA.raw • AA refers to ammonium acetate • Numbers refer to concentration of ammonium acetateFolder name: Excess CK2beta • Raw data of native mass spectrometry experiments with excess CK2β • E.g. CK2beta-0.20AA.raw • AA refers to ammonium acetate • Numbers refer to concentration of ammonium acetateFolder name: HDX • Processed HDX data using CK2α as search sequenceFolder name: Ion mobility • Raw data of ion mobility experiments • E.g. AH-H7.raw • First identifier (e.g. AH) refers to protein • Second identifier (e.g. H7) refers to wave height • AH = alcohol dehydrogenase • AVD = avidin • CCA = concanavalin A • PK = pyruvate kinaseThis work was supported by the Wellcome Trust [Wellcome Trust Strategic Award (090340/Z/09/Z)]

Research Data Supporting "Achieving small–molecule disruption of the CK2α/CK2β protein–protein interaction within the protein kinase CK2 heterotetramer"

Images of 1) isothermic traces of peptides, 2) compound structures, 3) native mass spectrometry, 4) competitive ITC trace, and 5) X-ray crystal structure of CK2a-NMR154 complexWellcome Trust (090340/Z/09/Z