I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.
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Nov 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
Abstract: Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.
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Nov 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548, 14043]
Abstract: Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å co-crystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared to the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein–protein interaction.
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Nov 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14794]
Open Access
Abstract: Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues.
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Nov 2020
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I04-Macromolecular Crystallography
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Open Access
Abstract: The SH2 domain‐containing protein of 76 kDa, SLP76, is an important adaptor protein that coordinates a complex protein network downstream of T‐cell receptors (TCR), ultimately regulating the immune response. Upon phosphorylation on Ser376, SLP76 interacts with 14‐3‐3 adaptor proteins, which leads to its proteolytic degradation. This provides a negative feedback mechanism by which TCR signalling can be controlled. To gain insight into the 14‐3‐3/SLP76 protein–protein interaction (PPI), we have determined a high‐resolution crystal structure of a SLP76 synthetic peptide containing Ser376 with 14‐3‐3σ. We then characterized its binding to 14‐3‐3 proteins biophysically by means of fluorescence polarization and isothermal titration calorimetry. Furthermore, we generated two recombinant SLP76 protein constructs and characterized their binding to 14‐3‐3. Our work lays the foundation for drug design efforts aimed at targeting the 14‐3‐3/SLP76 interaction and, thereby, TCR signalling.
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Nov 2020
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I04-Macromolecular Crystallography
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Torkild
Visnes
,
Carlos
Benítez-buelga
,
Armando
Cázares-körner
,
Kumar
Sanjiv
,
Bishoy M. F.
Hanna
,
Oliver
Mortusewicz
,
Varshni
Rajagopal
,
Julian J.
Albers
,
Daniel W
Hagey
,
Tove
Bekkhus
,
Saeed
Eshtad
,
Juan Miguel
Baquero
,
Geoffrey
Masuyer
,
Olov
Wallner
,
Sarah
Müller
,
Therese
Pham
,
Camilla
Göktürk
,
Azita
Rasti
,
Sharda
Suman
,
Raúl
Torres-ruiz
,
Antonio
Sarno
,
Elisée
Wiita
,
Evert J.
Homan
,
Stella
Karsten
,
Karthick
Marimuthu
,
Maurice
Michel
,
Tobias
Koolmeister
,
Martin
Scobie
,
Olga
Loseva
,
Ingrid
Almlöf
,
Judith Edda
Unterlass
,
Aleksandra
Pettke
,
Johan
Boström
,
Monica
Pandey
,
Helge
Gad
,
Patrick
Herr
,
Ann-sofie
Jemth
,
Samir
El andaloussi
,
Christina
Kalderén
,
Sandra
Rodriguez-perales
,
Javier
Benítez
,
Hans E
Krokan
,
Mikael
Altun
,
Pal
Stenmark
,
Ulrika Warpman
Berglund
,
Thomas
Helleday
Diamond Proposal Number(s):
[15806]
Open Access
Abstract: Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
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Nov 2020
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Yousuke
Yamada
,
Hajime
Takashima
,
David Lee
Walmsley
,
Fumihito
Ushiyama
,
Yohei
Matsuda
,
Harumi
Kanazawa
,
Toru
Yamaguchi-sasaki
,
Nozomi
Tanaka-yamamoto
,
Junya
Yamagishi
,
Risa
Kurimoto-tsuruta
,
Yuya
Ogata
,
Norikazu
Ohtake
,
Hayley
Angove
,
Lisa
Baker
,
Richard
Harris
,
Alba
Macias
,
Alan
Robertson
,
Allan
Surgenor
,
Hayato
Watanabe
,
Koichiro
Nakano
,
Masashi
Mima
,
Kunihiko
Iwamoto
,
Atsushi
Okada
,
Iichiro
Takata
,
Kosuke
Hitaka
,
Akihiro
Tanaka
,
Kiyoko
Fujita
,
Hiroyuki
Sugiyama
,
Roderick E.
Hubbard
Diamond Proposal Number(s):
[12428]
Abstract: UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation. A series was evolved from a fragment where a glycine moiety complexes zinc, which achieved low nanomolar potency in an enzyme functional assay but poor antibacterial activity on cell cultures. A second series was based on a fragment that chelated zinc through an imidazole moiety. Structure-guided design led to a 2-(1S-hydroxyethyl)-imidazole derivative exhibiting low nanomolar inhibition of LpxC and a minimum inhibitory concentration (MIC) of 4 μg/mL against Pseudomonas aeruginosa, which is little affected by the presence of albumin.
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Nov 2020
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I04-Macromolecular Crystallography
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Open Access
Abstract: Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.
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Nov 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lukasz F.
Sobala
,
Pearl Z.
Fernandes
,
Zalihe
Hakki
,
Andrew J.
Thompson
,
Jonathon D.
Howe
,
Michelle
Hill
,
Nicole
Zitzmann
,
Scott
Davies
,
Zania
Stamataki
,
Terry D.
Butters
,
Dominic S.
Alonzi
,
Spencer J.
Williams
,
Gideon
Davies
Diamond Proposal Number(s):
[1221, 12587, 18598]
Open Access
Abstract: Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.
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Nov 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Abstract: Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail a3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located > 40 Å apart in the same monomer but adjacent in two neighbouring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in inter-subunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.
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Nov 2020
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