I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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John
Liddle
,
Andrew C.
Pearce
,
Christopher
Arico-Muendel
,
Svetlana
Belyanskaya
,
Andrew
Brewster
,
Murray
Brown
,
Chun-Wa
Chung
,
Alexis
Denis
,
Nerina
Dodic
,
Anthony
Dossang
,
Peter
Eddershaw
,
Diana
Klimaszewska
,
Imran
Haq
,
Duncan S.
Holmes
,
Alistair
Jagger
,
Toral
Jakhria
,
Emilie
Jigorel
,
Ken
Lind
,
Jeff
Messer
,
Margaret
Neu
,
Allison
Olszewski
,
Riccardo
Ronzoni
,
James
Rowedder
,
Martin
Rüdiger
,
Steve
Skinner
,
Kathrine J.
Smith
,
Lionel
Trottet
,
Iain
Uings
,
Zhengrong
Zhu
,
James A.
Irving
,
David A.
Lomas
Diamond Proposal Number(s):
[23853, 17201]
Abstract: α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.
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Jun 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Julien
Orlans
,
Carole
Vincent-Monegat
,
Isabelle
Rahioui
,
Catherine
Sivignon
,
Agata
Butryn
,
Laurent
Soulère
,
Anna
Zaidman-Remy
,
Allen M.
Orville
,
Abdelaziz
Heddi
,
Pierre
Aller
,
Pedro
Da Silva
Diamond Proposal Number(s):
[19737, 19458, 23571]
Open Access
Abstract: Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.
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May 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620, 17221]
Open Access
Abstract: Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric‐acid xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single point mutation, G411V, trapping the protein in the inward facing conformation. Here we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesize that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1.
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May 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Human prolyl‐hydroxylases (PHDs) are hypoxia‐sensing 2‐oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia‐inducible factor target genes. PHD inhibition enables the treatment of anaemia/ischaemia‐related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late‐stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain‐penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active‐site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π‐π‐stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking β2–β3, which are involved in dynamic substrate binding/product release.
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Apr 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12342, 23269]
Open Access
Abstract: The rational design of linear peptides that assemble controllably and predictably in water is challenging. Short sequences must encode unique target structures and avoid alternative states. However, the non-covalent forces that stabilize and discriminate between states are weak. Nonetheless, for α-helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger α-helical barrels. Here, we show that pentameric to nonameric barrels are accessed by varying the residue at one of the h sites. In peptides with four L/I–K–E–I–A–x–Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting α-helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.
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Apr 2021
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I04-Macromolecular Crystallography
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Abstract: Here we have described a systematic structure activity relationship (SAR) of a set of compounds inspired from cladosporin, a tool compound that targets parasite (Plasmodium falciparum) lysyl tRNA synthetase (KRS). Four sets of analogues, synthesized based on point changes in the chemical scaffold of cladosporin and other logical modifications and hybridizations, were assessed using high throughput enzymatic and parasitic assays along with in vitro pharmacokinetics. Co-crystallization of the most potent compound in our series (CL-2) with PfKRS revealed its structural basis of enzymatic binding and potency. Further, we report that CL-2 has performed better than cladosporin in terms of metabolic stability. It thus represents a new lead for further optimization toward the development of antimalarial drugs. Collectively, along with a lead compound, the series offers insights on how even the slightest chemical modification might play an important role in enhancing or decreasing the potency of a chemical scaffold.
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Apr 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Von Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.
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Apr 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: Aspartate/asparagine‐β‐hydroxylase (AspH) is a human 2‐oxoglutarate (2OG) and Fe(II) oxygenase that catalyzes C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor‐like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate Fe(II) rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its Fe(II) binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variant retain substantial catalytic activity. Crystal structures clearly reveal metal‐ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non‐protein biomimetic catalysts.
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Apr 2021
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I04-Macromolecular Crystallography
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Hermen S.
Overkleeft
,
Sybrin
Schröder
,
Wendy
Offen
,
Alexandra
Males
,
Yi
Jin
,
Casper
De Boer
,
Jacopo
Enotarpi
,
Gijs
Van Der Marel
,
Bogdan
Florea
,
Jeroen
Codée
,
Gideon
Davies
Diamond Proposal Number(s):
[13587, 18598]
Abstract: There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate‐active enzymes. Activity‐based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here we show that non‐hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. We find that incorporation of carbasugar moieties, which we accomplished by cuprate‐assisted regioselective trans‐diaxial epoxide opening of carba‐mannal we synthesised for this purpose, yields inactivators that act as powerful activity‐based inhibitors for a‐1,6 endo‐mannanases. 3‐D structures at 1.35 – 1.47 Å resolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity‐based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families.
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Apr 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: ADAMTS13 is a plasma metalloprotease that is essential for the regulation of von Willebrand factor (VWF) function, mediator of platelet recruitment to sites of blood vessel damage. ADAMTS13 function is dynamically regulated by structural changes induced by VWF binding that convert it from a latent to active conformation. ADAMTS13 global latency is manifest by the interaction of its C-terminal CUB1-2 domains with its central Spacer domain. We resolved the crystal structure of the ADAMTS13 CUB1-2 domains revealing a previously unreported configuration for the tandem CUB domains. Docking simulations between the CUB1-2 domains with the Spacer domain in combination with enzyme kinetic functional characterization of ADAMTS13 CUB domain mutants enabled the mapping of the CUB1-2 domain site that binds the Spacer domain. Together, these data reveal the molecular basis of the ADAMTS13 Spacer-CUB interaction and the control of ADAMTS13 global latency.
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Apr 2021
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