I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Sergio
Celis
,
Fruzsina
Hobor
,
Thomas
James
,
Gail J.
Bartlett
,
Amaurys A.
Ibarra
,
Deborah K.
Shoemark
,
Zsofia
Hegedus
,
Kristina
Hetherington
,
Derek N.
Woolfson
,
Richard B.
Sessions
,
Thomas A.
Edwards
,
David M.
Andrews
,
Adam
Nelson
,
Andrew J.
Wilson
Diamond Proposal Number(s):
[19248]
Open Access
Abstract: Protein–protein interactions (PPIs) are central to biological mechanisms, and can serve as compelling targets for drug discovery. Yet, the discovery of small molecule inhibitors of PPIs remains challenging given the large and typically shallow topography of the interacting protein surfaces. Here, we describe a general approach to the discovery of orthosteric PPI inhibitors that mimic specific secondary protein structures. Initially, hot residues at protein–protein interfaces are identified in silico or from experimental data, and incorporated into secondary structure-based queries. Virtual libraries of small molecules are then shape-matched against the queries, and promising ligands docked to target proteins. The approach is exemplified experimentally using two unrelated PPIs that are mediated by an α-helix (p53/hDM2) and a β-strand (GKAP/SHANK1-PDZ). In each case, selective PPI inhibitors are discovered with low μM activity as determined by a combination of fluorescence anisotropy and 1H–15N HSQC experiments. In addition, hit expansion yields a series of PPI inhibitors with defined structure–activity relationships. It is envisaged that the generality of the approach will enable discovery of inhibitors of a wide range of unrelated secondary structure-mediated PPIs.
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Mar 2021
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I19-Small Molecule Single Crystal Diffraction
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Aaron J
Scott
,
Euan K.
Brechin
,
Stergios
Piligkos
,
Gopalan
Rajaraman
,
Mark
Murrie
,
Simon
Coles
,
Wim T.
Klooster
,
Gary S.
Nichol
,
Paul
Lusby
,
Lucy
Smythe
,
Arup
Sarkar
,
Julia
Vallejo
,
Emma
Regincos Marti
Diamond Proposal Number(s):
[22240]
Open Access
Abstract: Reaction of Ni(OTf)2 with the bisbidentate quaterpyridine ligand L results in the self-assembly of a tetrahedral, paramagnetic cage [NiII4L6]8+. By selectively exchanging the bound triflate from [OTf⊂NiII4L6](OTf)7 (1), we have been able to prepare a series of host-guest complexes that feauture an encapsulated paramagnetic tetrahalometallate ion inside this paramagnetic host giving [MIIX4⊂NiII4L6](OTf)6, where MIIX42− = MnCl42− (2), CoCl42− (5), CoBr42− (6), NiCl42− (7), CuBr42− (8) or [MIIIX4⊂NiII4L6](OTf)7, where MIIIX4− = FeCl4− (3), FeBr4− (4). Triflate-to-tetrahalometallate exchange occurs in solution and can also be accomplished through single-crystal-to-single-crystal transformations. Host-guest complexes 1-8 all crystallise as homochiral racemates in monoclinic space groups, wherein the four {NiN6} vertex within a single Ni4L6 unit possess the same Δ or Λ stereochemistry. Magnetic susceptibility and magnetisation data show that the magnetic exchange between metal ions in the host [NiII4] complex, and between the host and the MX4n- guest, are of comparable magnitude and antiferromagnetic in nature. Theoretically derived values for the magnetic exchange are in close agreement with experiment, revealing that large spin densities on the electronegative X-atoms of particular {MX4}n− guest molecules leads to stronger host-guest magnetic exchange interactions.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Husam Sabah
Auhim
,
Bella L.
Grigorenko
,
Tessa K.
Harris
,
Ozan E.
Aksakal
,
Igor V.
Polyakov
,
Colin
Berry
,
Gabriel Dos Passos
Gomes
,
Igor V.
Alabugin
,
Pierre J.
Rizkallah
,
Alexander V.
Nemukhin
,
D. Dafydd
Jones
Diamond Proposal Number(s):
[18812]
Open Access
Abstract: Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O2 located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation so forming a channel that allows O2 access to the immature chromophore. Absorbance spectroscopy supported by QM/MM simulations suggests that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation and the mechanistic insights open up new approaches for engineering optically controlled fluorescent proteins.
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Mar 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: 2-Oxoglutarate (2OG) is involved in biological processes including oxidations catalyzed by 2OG oxygenases for which it is a cosubstrate. Eukaryotic 2OG oxygenases have roles in collagen biosynthesis, lipid metabolism, DNA/RNA modification, transcriptional regulation, and the hypoxic response. Aspartate/asparagine-β-hydroxylase (AspH) is a human 2OG oxygenase catalyzing post-translational hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs) in the endoplasmic reticulum. AspH is of chemical interest, because its Fe(II) cofactor is complexed by two rather than the typical three residues. AspH is upregulated in hypoxia and is a prognostic marker on the surface of cancer cells. We describe studies on how derivatives of its natural 2OG cosubstrate modulate AspH activity. An efficient synthesis of C3- and/or C4-substituted 2OG derivatives, proceeding via cyanosulfur ylid intermediates, is reported. Mass spectrometry-based AspH assays with >30 2OG derivatives reveal that some efficiently inhibit AspH via competing with 2OG as evidenced by crystallographic and solution analyses. Other 2OG derivatives can substitute for 2OG enabling substrate hydroxylation. The results show that subtle changes, e.g. methyl- to ethyl-substitution, can significantly alter the balance between catalysis and inhibition. 3-Methyl-2OG, a natural product present in human nutrition, was the most efficient alternative cosubstrate identified; crystallographic analyses reveal the binding mode of (R)-3-methyl-2OG and other 2OG derivatives to AspH and inform on the balance between turnover and inhibition. The results will enable the use of 2OG derivatives as mechanistic probes for other 2OG utilizing enzymes and suggest 2-oxoacids other than 2OG may be employed by some 2OG oxygenases in vivo.
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Feb 2021
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I11-High Resolution Powder Diffraction
I12-JEEP: Joint Engineering, Environmental and Processing
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Kieran W. P.
Orr
,
Sean M.
Collins
,
Emily M.
Reynolds
,
Frank
Nightingale
,
Hanna L. B.
Bostroem
,
Simon J.
Cassidy
,
Daniel M.
Dawson
,
Sharon E.
Ashbrook
,
Oxana
Magdysyuk
,
Paul A.
Midgley
,
Andrew L.
Goodwin
,
Hamish H.-m.
Yeung
Diamond Proposal Number(s):
[20946, 18786]
Open Access
Abstract: Control over the spatial distribution of components in metal–organic frameworks has potential to unlock improved performance and new behaviour in separations, sensing and catalysis. We report an unprecedented single-step synthesis of multi-component metal–organic framework (MOF) nanoparticles based on the canonical ZIF-8 (Zn) system and its Cd analogue, which form with a core–shell structure whose internal interface can be systematically tuned. We use scanning transmission electron microscopy, X-ray energy dispersive spectroscopy and a new composition gradient model to fit high-resolution X-ray diffraction data to show how core–shell composition and interface characteristics are intricately controlled by synthesis temperature and reaction composition. Particle formation is investigated by in situ X-ray diffraction, which reveals that the spatial distribution of components evolves with time and is determined by the interplay of phase stability, crystallisation kinetics and diffusion. This work opens up new possibilities for the control and characterisation of functionality, component distribution and interfaces in MOF-based materials.
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Feb 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Maria-agustina
Rossi
,
Veronica
Martinez
,
Philip
Hinchliffe
,
Maria F.
Mojica
,
Valerie
Castillo
,
Diego M.
Moreno
,
Ryan
Smith
,
Brad
Spellberg
,
George L.
Drusano
,
Claudia
Banchio
,
Robert A.
Bonomo
,
James
Spencer
,
Alejandro J.
Vila
,
Graciela
Mahler
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-β-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all β-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., Ki = 0.44 μM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(II) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than D/L-captopril. Unexpectedly, MMTZ binding features a thioether–π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur–π interactions can be exploited for general ligand design in medicinal chemistry.
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Jan 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19248]
Open Access
Abstract: β-Strand mediated protein–protein interactions (PPIs) represent underexploited targets for chemical probe development despite representing a significant proportion of known and therapeutically relevant PPI targets. β-Strand mimicry is challenging given that both amino acid side-chains and backbone hydrogen-bonds are typically required for molecular recognition, yet these are oriented along perpendicular vectors. This paper describes an alternative approach, using GKAP/SHANK1 PDZ as a model and dynamic ligation screening to identify small-molecule replacements for tranches of peptide sequence. A peptide truncation of GKAP functionalized at the N- and C-termini with acylhydrazone groups was used as an anchor. Reversible acylhydrazone bond exchange with a library of aldehyde fragments in the presence of the protein as template and in situ screening using a fluorescence anisotropy (FA) assay identified peptide hybrid hits with comparable affinity to the GKAP peptide binding sequence. Identified hits were validated using FA, ITC, NMR and X-ray crystallography to confirm selective inhibition of the target PDZ-mediated PPI and mode of binding. These analyses together with molecular dynamics simulations demonstrated the ligands make transient interactions with an unoccupied basic patch through electrostatic interactions, establishing proof-of-concept that this unbiased approach to ligand discovery represents a powerful addition to the armory of tools that can be used to identify PPI modulators.
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Jan 2021
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I11-High Resolution Powder Diffraction
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Diamond Proposal Number(s):
[21755, 8786]
Open Access
Abstract: We report four new A-site vacancy ordered thiocyanate double double perovskites,A1–x{Ni[Bi(SCN)6](1–x)/3}, A = K+, NH4+, CH3(NH3)+ (MeNH3+) and C(NH2)3+ (Gua+), includingthe first examples of thiocyanate perovskites containing organic A-site cations. We show, usinga combination of X-ray and neutron diffraction, that the structure of these frameworks dependson the A-site cation, and that these frameworks possess complex vacancy-ordering patterns andcooperative octahedral tilts distinctly different from atomic perovskites. Density functional theorycalculations uncover the energetic origin of these complex orders and allow us to propose asimple rule to predict favoured A-site cation orderings for a given tilt sequence. We use theseinsights, in combination with symmetry mode analyses, to show that these complex orders offera new route to non-centrosymmetric perovskites which render them as excellent candidates forpiezo- and ferroelectric applications.
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Jan 2021
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B18-Core EXAFS
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Diamond Proposal Number(s):
[20856]
Open Access
Abstract: The catalytic synthesis of NH3 from the thermodynamically challenging N2 reduction reaction under mild conditions is currently a significant problem for scientists. Accordingly, herein, we report the development of a nitrogenase-inspired inorganic-based chalcogenide system for the efficient electrochemical conversion of N2 to NH3, which is comprised of the basic structure of [Fe–S2–Mo]. This material showed high activity of 8.7 mgNH3 mgFe−1 h−1 (24 μgNH3 cm−2 h−1) with an excellent faradaic efficiency of 27% for the conversion of N2 to NH3 in aqueous medium. It was demonstrated that the Fe1 single atom on [Fe–S2–Mo] under the optimal negative potential favors the reduction of N2 to NH3 over the competitive proton reduction to H2. Operando X-ray absorption and simulations combined with theoretical DFT calculations provided the first and important insights on the particular electron-mediating and catalytic roles of the [Fe–S2–Mo] motifs and Fe1, respectively, on this two-dimensional (2D) molecular layer slab.
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Jan 2021
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[23480]
Open Access
Abstract: The length and constitution of spacers linking three 2,6-pyridinedicarboxamide units in a molecular strand influence the tightness of the resulting overhand (open-trefoil) knot that the strand folds into in the presence of lanthanide(III) ions. The use of β-hairpin forming motifs as linkers enables a metal-coordinated pseudopeptide with a knotted tertiary structure to be generated. The resulting pseudopeptide knot has one of the highest backbone-to-crossing ratios (BCR)—a measure of knot tightness (a high value corresponding to looseness)—for a synthetic molecular knot to date. Preorganization in the crossing-free turn section of the knot affects aromatic stacking interactions close to the crossing region. The metal-coordinated pseudopeptide knot is compared to overhand knots with other linkers of varying tightness and turn preorganization, and the entangled architectures characterized by NMR spectroscopy, ESI-MS, CD spectroscopy and, in one case, X-ray crystallography. The results show how it is possible to program specific conformational properties into different key regions of synthetic molecular knots, opening the way to systems where knotting can be systematically incorporated into peptide-like chains through design.
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Dec 2020
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