I03-Macromolecular Crystallography
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Samuel L.
Freeman
,
Vera
Skafar
,
Hanna
Kwon
,
Alistair J.
Fielding
,
Peter C. E.
Moody
,
Alejandra
Martínez
,
Federico
Issoglio
,
Lucas
Inchausti
,
Pablo
Smircich
,
Ari
Zeida
,
Lucía
Piacenza
,
Rafael
Radi
,
Emma L.
Raven
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defence systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance (EPR) spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] Compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c, but is still competent for oxididation of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.
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Jun 2022
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I04-Macromolecular Crystallography
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Pamela
Sweeney
,
Ashleigh
Galliford
,
Abhishek
Kumar
,
Dinesh
Raju
,
Naveen B.
Krishna
,
Emmajay
Sutherland
,
Caitlin J.
Leo
,
Gemma
Fisher
,
Roopa
Lalitha
,
Likith
Muthuraj
,
Gladstone
Sigamani
,
Verena
Oehler
,
Silvia
Synowsky
,
Sally L.
Shirran
,
Tracey M.
Gloster
,
Clarissa M.
Czekster
,
Pravin
Kumar
,
Rafael G.
Da Silva
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we characterize the structure and function of S. aureus TrmK using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the S. aureus TrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for S. aureus TrmK-catalysed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In-silico screening of compounds suggested the multi-functional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of S. aureus TrmK, laying a foundation for future structure-based drug discovery.
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May 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8035]
Open Access
Abstract: Thrombospondin Type-1 Repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding, and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization is poorly understood. Here we conducted molecular dynamics simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.
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May 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: The Candidate Phyla Radiation is a recently uncovered and vast expansion of the bacterial domain of life, made up of largely uncharacterized phyla that lack isolated representatives. This unexplored territory of genetic diversity presents an abundance of novel proteins with potential applications in the life-science sectors. Here we present the structural and functional elucidation of CPR-C4, a hypothetical protein from the genome of a thermophilic Candidate Phyla Radiation organism, identified through metagenomic sequencing. Our analyses revealed that CPR-C4 is a member of a family of highly conserved proteins within the Candidate Phyla Radiation. The function of CPR-C4 as a cysteine protease was predicted through remote structural similarity to the Homo sapiens vasohibins and subsequently confirmed experimentally with fluorescence-based activity assays. Furthermore, detailed structural and sequence alignment analysis enabled identification of a non-canonical cysteine-histidine-leucine(carbonyl) catalytic triad. The unexpected structural and functional similarities between CPR-C4 and the human vasohibins suggest an evolutionary relationship undetectable at the sequence level alone.
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Apr 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Alice R.
Cross
,
Sumita
Roy
,
Mirella
Vivoli Vega
,
Martin
Rejzek
,
Sergey A.
Nepogodiev
,
Matthew
Cliff
,
Debbie
Salmon
,
Michail N.
Isupov
,
Robert A.
Field
,
Joann L.
Prior
,
Nicholas J.
Harmer
Diamond Proposal Number(s):
[16378]
Open Access
Abstract: The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, whilst DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologues of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologues in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3’’,5’’ double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3’’-position than the 5’’-position, in contrast to a previously studied orthologue. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP, and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralogue most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.
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Apr 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells, and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound ISFP1 (Isothiazolone Fragment of PGM) as targeting a cysteine residue not conserved in the human orthologue. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC50 of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.
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Apr 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[12788]
Open Access
Abstract: The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide (prFMN) cofactor for catalysis. However, only a handful of UbiD family members have been characterised in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism, linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering (SAXS) measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO2 under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.
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Feb 2022
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[18074]
Open Access
Abstract: Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation and ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. Recent high-resolution cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, and channel residues are likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the cryo-EM structure of mouse complex I with an extremely tight-binding natural-product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties ideally spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound, but stabilises the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.
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Jan 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical re-engineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent post-cleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent pre-cleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the post-cleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q and E493K confirmed the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.
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Dec 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as anti-tumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. Recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2/UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. Solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and supporting NMR data will facilitate rational re-design of small molecules that could evade AGP and therefore improve tissue distribution.
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Nov 2021
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