Talos-Talos Arctica at Diamond
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Paul B.
Klar
,
David G.
Waterman
,
Tim
Gruene
,
Debakshi
Mullick
,
Yun
Song
,
James B.
Gilchrist
,
C. David
Owen
,
Wen
Wen
,
Idan
Biran
,
Lothar
Houben
,
Neta
Regev-Rudzki
,
Ron
Dzikowski
,
Noa
Marom
,
Lukas
Palatinus
,
Peijun
Zhang
,
Leslie
Leiserowitz
,
Michael
Elbaum
Diamond Proposal Number(s):
[21004, 29812]
Open Access
Abstract: Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development.
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Jul 2024
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I03-Macromolecular Crystallography
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Francesca
Nonne
,
Lucia
Dello Iacono
,
Sara
Bertuzzi
,
Luca
Unione
,
Daniela
Proietti
,
Nathalie
Norais
,
Immaculada
Margarit
,
Roberto
Adamo
,
Jesús
Jiménez-Barbero
,
Filippo
Carboni
,
Maria Rosaria
Romano
Open Access
Abstract: Glycoconjugate vaccines so far licensed are generally composed of a native or size-reduced capsular polysaccharide conjugated to carrier proteins. Detailed information on the structural requirements necessary for CPS recognition is becoming the key to accelerating the development of next-generation improved glycoconjugate vaccines. Structural glycobiology studies using oligosaccharides (OS) complexed with functional monoclonal antibodies represent a powerful tool for gaining information on CPS immunological determinants at the atomic level. Herein, the minimal structural epitope of Haemophilus influenzae type b (Hib) CPS recognized by a functional human monoclonal antibody (hmAb) is reported. Short and well-defined Hib oligosaccharides originating from the depolymerization of the native CPS have been used to elucidate saccharide–mAb interactions by using a multidisciplinary approach combining surface plasmon resonance (SPR), saturation transfer difference-nanomagnetic resonance (STD-NMR), and X-ray crystallography. Our study demonstrates that the minimal structural epitope of Hib is comprised within two repeating units (RUs) where ribose and ribitol are directly engaged in the hmAb interaction, and the binding pocket fully accommodates two RUs without any additional involvement of a third one. Understanding saccharide antigen structural characteristics can provide the basis for the design of innovative glycoconjugate vaccines based on alternative technologies, such as synthetic or enzymatic approaches.
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Feb 2024
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I03-Macromolecular Crystallography
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Qi
Zhou
,
Pablo
Catalán
,
Helen
Bell
,
Patrick
Baumann
,
Rebekah
Cooke
,
Rhodri
Evans
,
Jianhua
Yang
,
Zhen
Zhang
,
Davide
Zappalà
,
Ye
Zhang
,
George Michael
Blackburn
,
Yuan
He
,
Yi
Jin
Diamond Proposal Number(s):
[20147]
Open Access
Abstract: Antibiotic-resistant Enterobacterales that produce oxacillinase (OXA)-48-like Class D β-lactamases are often linked to increased clinical mortality. Though the catalytic mechanism of OXA-48 is known, the molecular origin of its biphasic kinetics has been elusive. We here identify selective chloride binding rather than decarbamylation of the carbamylated lysine as the source of biphasic kinetics, utilizing isothermal titration calorimetry (ITC) to monitor the complete reaction course with the OXA-48 variant having a chemically stable N-acetyl lysine. Further structural investigation enables us to capture an unprecedented inactive acyl intermediate wedged in place by a halide ion paired with a conserved active site arginine. Supported by mutagenesis and mathematical simulation, we identify chloride as a “Janus effector” that operates by allosteric activation of the burst phase and by inhibition of the steady state in kinetic assays of β-lactams. We show that chloride-induced biphasic kinetics directly affects antibiotic efficacy and facilitates the differentiation of clinical isolates encoding Class D from Class A and B carbapenemases. As chloride is present in laboratory and clinical procedures, our discovery greatly expands the roles of chloride in modulating enzyme catalysis and highlights its potential impact on the pharmacokinetics and efficacy of antibiotics during in vivo treatment.
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Dec 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[24948, 18598]
Open Access
Abstract: Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a “low-tech”, easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.
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Nov 2023
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I03-Macromolecular Crystallography
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Dyana N.
Kenanova
,
Emira
Visser
,
Johanna M.
Virta
,
Eline
Sijbesma
,
Federica
Centorrino
,
Holly R.
Vickery
,
Mengqi
Zhong
,
R. Jeffrey
Neitz
,
Luc
Brunsveld
,
Christian
Ottmann
,
Michelle R.
Arkin
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Dysregulation of protein–protein interactions (PPIs) commonly leads to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for identifying reversibly covalent small molecules. We explored the scope of disulfide tethering for the discovery of selective PPI stabilizers (molecular glues) using the hub protein 14-3-3σ. We screened complexes of 14-3-3 with 5 biologically and structurally diverse phosphopeptides derived from the 14-3-3 client proteins ERα, FOXO1, C-RAF, USP8, and SOS1. Stabilizing fragments were found for 4/5 client complexes. Structural elucidation of these complexes revealed the ability of some peptides to conformationally adapt to make productive interactions with the tethered fragments. We validated eight fragment stabilizers, six of which showed selectivity for one phosphopeptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious fragment increased 14-3-3σ/C-RAF phosphopeptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3σ provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.
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Apr 2023
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Krios II-Titan Krios II at Diamond
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Yasunori
Watanabe
,
Luiza
Mendonca
,
Elizabeth R.
Allen
,
Andrew
Howe
,
Mercede
Lee
,
Joel D.
Allen
,
Himanshi
Chawla
,
David
Pulido
,
Francesca
Donnellan
,
Hannah
Davies
,
Marta
Ulaszewska
,
Sandra
Belij-Rammerstorfer
,
Susan
Morris
,
Anna-Sophia
Krebs
,
Wanwisa
Dejnirattisai
,
Juthathip
Mongkolsapaya
,
Piyada
Supasa
,
Gavin R.
Screaton
,
Catherine M.
Green
,
Teresa
Lambe
,
Peijun
Zhang
,
Sarah C.
Gilbert
,
Max
Crispin
Diamond Proposal Number(s):
[18477, 21005, 21004]
Open Access
Abstract: Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation, and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirm the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.
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Apr 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Mahima
Sharma
,
Palika
Abayakoon
,
Ruwan
Epa
,
Yi
Jin
,
James P.
Lingford
,
Tomohiro
Shimada
,
Masahiro
Nakano
,
Janice W.-Y.
Mui
,
Akira
Ishihama
,
Ethan D.
Goddard-Borger
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[13587, 18598, 24948]
Open Access
Abstract: The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on Earth and is metabolized by bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden–Meyerhof–Parnas pathway metabolizes SQ to produce dihydroxyacetone phosphate and sulfolactaldehyde and is analogous to the classical Embden–Meyerhof–Parnas glycolysis pathway for the metabolism of glucose-6-phosphate, though the former only provides one C3 fragment to central metabolism, with excretion of the other C3 fragment as dihydroxypropanesulfonate. Here, we report a comprehensive structural and biochemical analysis of the three core steps of sulfoglycolysis catalyzed by SQ isomerase, sulfofructose (SF) kinase, and sulfofructose-1-phosphate (SFP) aldolase. Our data show that despite the superficial similarity of this pathway to glycolysis, the sulfoglycolytic enzymes are specific for SQ metabolites and are not catalytically active on related metabolites from glycolytic pathways. This observation is rationalized by three-dimensional structures of each enzyme, which reveal the presence of conserved sulfonate binding pockets. We show that SQ isomerase acts preferentially on the β-anomer of SQ and reversibly produces both SF and sulforhamnose (SR), a previously unknown sugar that acts as a derepressor for the transcriptional repressor CsqR that regulates SQ-utilization. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex modulation by the metabolites SQ, SLA, AMP, ADP, ATP, F6P, FBP, PEP, DHAP, and citrate, and we show that SFP aldolase reversibly synthesizes SFP. This body of work provides fresh insights into the mechanism, specificity, and regulation of sulfoglycolysis and has important implications for understanding how this biochemistry interfaces with central metabolism in prokaryotes to process this major repository of biogeochemical sulfur.
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Feb 2021
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[22214]
Open Access
Abstract: The exploration of complex multicomponent chemical reactions leading to new clusters, where discovery requires both molecular self-assembly and crystallization, is a major challenge. This is because the systematic approach required for an experimental search is limited when the number of parameters in a chemical space becomes too large, restricting both exploration and reproducibility. Herein, we present a synthetic strategy to systematically search a very large set of potential reactions, using an inexpensive, high-throughput platform that is modular in terms of both hardware and software and is capable of running multiple reactions with in-line analysis, for the automation of inorganic and materials chemistry. The platform has been used to explore several inorganic chemical spaces to discover new and reproduce known tungsten-based, mixed transition-metal polyoxometalate clusters, giving a digital code that allows the easy repeat synthesis of the clusters. Among the many species identified in this work, the most significant is the discovery of a novel, purely inorganic W24FeIII–superoxide cluster formed under ambient conditions. The modular wheel platform was employed to undertake two chemical space explorations, producing compounds 1–4: (C2H8N)10Na2[H6Fe(O2)W24O82] (1, {W24Fe}), (C2H8N)72Na16[H16Co8W200O660(H2O)40] (2, {W200Co8}), (C2H8N)72Na16[H16Ni8W200O660(H2O)40] (3, {W200Ni8}), and (C2H8N)14[H26W34V4O130] (4, {W34V4}), along with many other known species, such as simple Keggin clusters and 1D {W11M2+} chains.
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Aug 2020
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Lukasz F.
Sobala
,
Gaetano
Speciale
,
Sha
Zhu
,
Lluı́s
Raich
,
Natalia
Sannikova
,
Andrew J.
Thompson
,
Zalihe
Hakki
,
Dan
Lu
,
Saeideh
Shamsi Kazem Abadi
,
Andrew R.
Lewis
,
Vı́ctor
Rojas-Cervellera
,
Ganeko
Bernardo-Seisdedos
,
Yongmin
Zhang
,
Oscar
Millet
,
Jesús
Jiménez-Barbero
,
Andrew J.
Bennet
,
Matthieu
Sollogoub
,
Carme
Rovira
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/KM) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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Apr 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Casper
De Boer
,
Nicholas G. S.
Mcgregor
,
Rhianna J.
Rowland
,
Olga
Moroz
,
Elena
Blagova
,
Jos
Reijngoud
,
Mark
Arentshorst
,
David
Osborn
,
Marc D.
Morant
,
Eric
Abbate
,
Mary A.
Stringer
,
Kristian B. R. M.
Krogh
,
Lluís
Raich
,
Carme
Rovira
,
Jean-Guy
Berrin
,
Gilles P.
Van Wezel
,
Arthur F. J.
Ram
,
Bogdan I.
Florea
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Keith S.
Wilson
,
Liang
Wu
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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May 2019
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