I02-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[8367]
Open Access
Abstract: N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These snapshot structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carboncarbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carboncarbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the CC bond-forming pathway will allow further development of these enzymes for stereospecific synthesis of new enzyme products.
|
Apr 2014
|
|
I02-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[7613]
Open Access
Abstract: DNA transposases catalyze the movement of transposons around genomes by a cut-and-paste mechanism related to retroviral integration. Transposases and retroviral integrases share a common RNaseH-like domain with a catalytic DDE/D triad that coordinates the divalent cations required for DNA cleavage and integration. The anti-retroviral drugs Raltegravir and Elvitegravir inhibit integrases by displacing viral DNA ends from the catalytic metal ions. We demonstrate that Raltegravir, but not Elvitegravir, binds to Mos1 transposase in the presence of Mg2+ or Mn2+, without the requirement for transposon DNA, and inhibits transposon cleavage and DNA integration in biochemical assays. Crystal structures at 1.7 Å resolution show Raltegravir, in common with integrases, coordinating two Mg2+ or Mn2+ ions in the Mos1 active site. However, in the absence of transposon ends, the drug adopts an unusual, compact binding mode distinct from that observed in the active site of the prototype foamy virus integrase.
|
Jan 2014
|
|
I02-Macromolecular Crystallography
|
Hyung-gu
Kim
,
Li
Tan
,
Ellen L.
Weisberg
,
Feiyang
Liu
,
Peter
Canning
,
Hwan Geun
Choi
,
Scott A.
Ezell
,
Hong
Wu
,
Zheng
Zhao
,
Jinhua
Wang
,
Anna
Mandinova
,
James D.
Griffin
,
Alex N.
Bullock
,
Qingsong
Liu
,
Sam W.
Lee
,
Nathanael S.
Gray
Open Access
Abstract: The DDR1 receptor tyrosine kinase is activated by matrix collagens and has been implicated in numerous cellular functions such as proliferation, differentiation, adhesion, migration, and invasion. Here we report the discovery of a potent and selective DDR1 inhibitor, DDR1-IN-1, and present the 2.2 Å DDR1 co-crystal structure. DDR1-IN-1 binds to DDR1 in the ‘DFG-out’ conformation and inhibits DDR1 autophosphorylation in cells at submicromolar concentrations with good selectivity as assessed against a panel of 451 kinases measured using the KinomeScan technology. We identified a mutation in the hinge region of DDR1, G707A, that confers >20-fold resistance to the ability of DDR1-IN-1 to inhibit DDR1 autophosphorylation and can be used to establish what pharmacology is DDR1-dependent. A combinatorial screen of DDR1-IN-1 with a library of annotated kinase inhibitors revealed that inhibitors of PI3K and mTOR such as GSK2126458 potentiate the antiproliferative activity of DDR1-IN-1 in colorectal cancer cell lines. DDR1-IN-1 provides a useful pharmacological probe for DDR1-dependent signal transduction.
|
Oct 2013
|
|
I24-Microfocus Macromolecular Crystallography
|
Michael D.
Urbaniak
,
Iain T.
Collie
,
Wenxia
Fang
,
Tonia
Aristotelous
,
Susanne
Eskilsson
,
Olawale G.
Raimi
,
Justin
Harrison
,
Iva Hopkins
Navratilova
,
Julie A.
Frearson
,
Daan M. F.
Van Aalten
,
Michael A. J.
Ferguson
Abstract: Bacteriocin; P aeruginosa.; pyocin S2; pyocin AP41; immunity protein Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi−bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP−inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP.
The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
|
Sep 2013
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[6388]
Abstract: Acetolactate decarboxylase catalyzes the conversion of both enantiomers of acetolactate to the (R)-enantiomer of acetoin, via a mechanism that has been shown to involve a prior rearrangement of the non-natural (R)-enantiomer substrate to the natural (S)-enantiomer. In this paper, a series of crystal structures of ALDC complex with designed transition state mimics are reported. These structures, coupled with inhibition studies and site-directed mutagenesis provide an improved understanding of the molecular processes involved in the stereoselective decarboxylation/protonation events. A mechanism for the transformation of each enantiomer of acetolactate is proposed.
|
Aug 2013
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[8359]
Abstract: Most protein kinases are regulated through activation loop phosphorylation, but the contributions of individual sites are largely unresolved due to insufficient control over sample phosphorylation. Aurora-A is a mitotic Ser/Thr protein kinase that has two regulatory phosphorylation sites on its activation loop, T287 and T288. While phosphorylation of T288 is known to activate the kinase, the function of T287 phosphorylation is unclear. We applied site-directed mutagenesis and selective chemical modification to specifically introduce bioisosteres for phospho-threonine and other unnatural amino acids at these positions. Modified Aurora-A proteins were characterized using a biochemical assay measuring substrate phosphorylation. Replacement of T288 with glutamate and aspartate weakly stimulated activity. Phospho-cysteine, installed by chemical synthesis from a corresponding cysteine residue introduced at position 288, showed catalytic activity approaching that of the comparable phospho-serine protein. Unnatural amino acid residues, with longer side chains, inserted at position 288 were autophosphorylated and supported substrate phosphorylation. Aurora-A activity is enhanced by phosphorylation at position 287 alone but is suppressed when position 288 is also phosphorylated. This is rationalized by competition between phosphorylated T287 and T288 for a binding site composed of arginines, based on a structure of Aurora-A in which phospho-T287 occupies this site. This is, to our knowledge, the first example of a Ser/Thr kinase whose activity is controlled by the phosphorylation state of adjacent residues in its activation loop. Overall we demonstrate an approach that combines mutagenesis and selective chemical modification of selected cysteine residues to investigate otherwise impenetrable aspects of kinase regulation.
|
Aug 2013
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Sander S.
Van Berkel
,
Joanne
Nettleship
,
Ivanhoe K. H.
Leung
,
Jurgen
Brem
,
Hwanho
Choi
,
David I.
Stuart
,
Timothy D. W.
Claridge
,
Michael A.
Mcdonough
,
Raymond J.
Owens
,
Jingshan
Ren
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[8423]
Abstract: â-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product.
|
Aug 2013
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[6388]
Open Access
Abstract: Recent evidence established that the cell envelope of Mycobacterium tuberculosis, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of M. tuberculosis TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients.
|
Jul 2013
|
|
I04-Macromolecular Crystallography
|
Anders E. G.
Lindgren
,
Tobias
Karlberg
,
Ann-gerd
Thorsell
,
Mareike
Hesse
,
Sara
Spjut
,
Torun
Ekblad
,
C. David
Andersson
,
Ana Filipa
Pinto
,
Johan
Weigelt
,
Michael O.
Hottiger
,
Anna
Linusson
,
Mikael
Elofsson
,
Herwig
Schüler
Diamond Proposal Number(s):
[6603]
Abstract: Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.
|
Jun 2013
|
|
I02-Macromolecular Crystallography
|
Rasheduzzaman
Chowdhury
,
José Ignacio
Candela-lena
,
Mun Chiang
Chan
,
David Jeremy
Greenald
,
Kar Kheng
Yeoh
,
Ya-min
Tian
,
Michael A.
Mcdonough
,
Anthony
Tumber
,
Nathan R.
Rose
,
Ana
Conejo-garcia
,
Marina
Demetriades
,
Sinnakaruppan
Mathavan
,
Akane
Kawamura
,
Myung Kyu
Lee
,
Freek
Van Eeden
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Christopher J.
Schofield
Open Access
Abstract: The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on thefunctional effects of PHD inhibition in cells and in animals.
|
May 2013
|
|
