I03-Macromolecular Crystallography
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David
Madariaga
,
Nuria
Martínez-sáez
,
Víctor J.
Somovilla
,
Helena
Coelho
,
Jessika
Valero-gonzález
,
Jorge
Castro-lópez
,
Juan L.
Asensio
,
Jesús
Jiménez-barbero
,
Jesús H.
Busto
,
Alberto
Avenoza
,
Filipa
Marcelo
,
Ramón
Hurtado-guerrero
,
Francisco
Corzana
,
Jesús M.
Peregrina
Diamond Proposal Number(s):
[8035]
Abstract: Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide–lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.
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Dec 2014
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9424]
Open Access
Abstract: Constrained alpha-helical peptides are an exciting class of molecule designed to disrupt protein-protein interactions (PPIs) at a surface-exposed helix binding site. Complexes that engage more than one helical face account for over a third of structurally characterized helix PPIs, including several examples where the helix is fully buried. However, no constrained peptides have been reported that have targeted this class of interaction. We report the design of stapled and hydrogen bond surrogate (HBS) peptides mimicking the helical tail of the malaria parasite invasion motor myosin (myoA), which presents polar and hydrophobic functionality on all three faces in binding its partner, myoA tail interacting protein (MTIP), with high affinity. The first structures of these different constrained peptides bound to the same target are reported, enabling a direct comparison between these constraints and between staples based on monosubstituted pentenyl glycine (pGly) and disubstituted pentenyl alanine (pAla). Importantly, installation of these constraints does not disrupt native interactions in the buried site, so the affinity of the wild-type peptide is maintained.
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Aug 2014
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8574]
Abstract: Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages.
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Aug 2014
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I02-Macromolecular Crystallography
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David
Albesa-jove
,
Laurent R.
Chiarelli
,
Vadim
Makarov
,
Maria Rosalia
Pasca
,
Saioa
Urresti
,
Giorgia
Mori
,
Elena
Salina
,
Anthony
Vocat
,
Natalia
Comino
,
Elisabeth
Mohorko
,
Svetlana
Ryabova
,
Bernhard
Pfieiffer
,
Ana Luisa De Jesus
Lopes Ribeiro
,
Ane
Rodrigo-unzueta
,
Montse
Tersa
,
Giuseppe
Zanoni
,
Silvia
Buroni
,
Karl-heinz
Altmann
,
Ruben C.
Hartkoorn
,
Rudi
Glockshuber
Diamond Proposal Number(s):
[8302]
Open Access
Abstract: The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 angstrom resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a beta-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti- mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.
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Jul 2014
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I02-Macromolecular Crystallography
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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.
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Apr 2014
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I02-Macromolecular Crystallography
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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.
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Jan 2014
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I02-Macromolecular Crystallography
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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.
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Oct 2013
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I24-Microfocus Macromolecular Crystallography
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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.
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Sep 2013
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I03-Macromolecular Crystallography
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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.
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Aug 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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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.
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Aug 2013
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