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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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ali
Nejatie
,
Elizabeth
Steves
,
Nick
Gauthier
,
Jamie
Baker
,
Jason
Nesbitt
,
Stephen A.
Mcmahon
,
Verena
Oehler
,
Nicholas J.
Thornton
,
Benjamin
Noyovitz
,
Kobra
Khazaei
,
Brock W.
Byers
,
Wesley F.
Zandberg
,
Tracey M.
Gloster
,
Margo M.
Moore
,
Andrew J.
Bennet
Abstract: Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus (AtS) and Trichophyton rubrum (TrS). Unlike A. fumigatus Kdnase (AfS), hydrolysis with the Neu5Ac substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AfS but 30-fold higher by TrS. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both AtS and TrS show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of AfS in vesicles at the cell surface.
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Nov 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Abstract: Viruses can infect all cellular organisms, from bacteria to humans. Once they have tricked their way into the cell, they exploit the host’s cellular machinery to make more copies of themselves, which can be released and go on to infect other cells. Organisms have developed strategies to recognise and defend against any virus infection, called adaptive immunity. However, viruses have evolved a
way to combat the host defence mechanism, meaning they ultimately win the infection battle.
Microbes use an adaptive immunity mechanism called CRISPR against virus infection. An international team of researchers focussed on one type of CRISPR (type III) and investigated how viruses overcome the microbial CRISPR defence system. Data they collected on the Microfocus Macromolecular Crystallography (MX) beamline I24 at Diamond Light Source provided vital insights into the mechanism used by viruses to overcome the microbial defence. Microbes with the type III CRISPR defence system produce a cyclic molecule in response to virus detection. It signals that there is an infection and kick-starts cellular processes to combat the attack. However, viruses have evolved an enzyme that binds to and destroys the cyclic molecule, neutralising the defence mechanism. This research used the structure of the viral enzyme, bound to the cyclic molecule, to reveal key details of how the viral enzyme recognises this molecule and how this translates into function. The results show the fundamental mechanism underlying how viruses out-manoeuvre microbe defences against infection. We may be able to use this understanding to engineer viruses to target drug-resistant bacteria that infect humans.
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Jul 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Abstract: The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD+:acetoacetate and NAD+:3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.
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Dec 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.
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Jun 2020
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19844, 21035]
Open Access
Abstract: The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Here we describe the structure and mechanism of a cOA-activated CRISPR defence DNA endonuclease, CRISPR ancillary nuclease 1 (Can1). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.
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Jan 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Abstract: The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3,4,5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7,8,9,10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host–pathogen interactions11,12,13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Marta
Artola
,
Chi-Lin
Kuo
,
Stephen
Mcmahon
,
Verena
Oehler
,
Thomas
Hansen
,
Martijn
Van der lienden
,
Xu
He
,
Hans
Van den elst
,
Bogdan I.
Florea
,
Allison R.
Kermode
,
Gijsbert A.
Van der marel
,
Tracey M.
Gloster
,
Jeroen D. C.
Codée
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity‐based glycosidase probes (ABPs). Direct 3‐amino‐2‐(trifluoromethyl)quinazolin‐4(3H)‐one‐mediated aziridination of l‐ido‐configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α‐l‐iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity‐based manner with human recombinant α‐l‐iduronidase (rIDUA, Aldurazyme®). The structures of IDUA when complexed with the inhibitors in a non‐covalent transition state mimicking form and a covalent enzyme‐bound form provide insights into its conformational itinerary. Inhibitors 1–3 adopt a half‐chair conformation in solution (4H3 and 3H4), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1–3 may need to overcome an energy barrier in order to switch from the 4H3 conformation to the transition state (2, 5B) binding conformation before reacting and adopting a covalent 5S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.
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Nov 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Weiwu
Ren
,
Robert
Pengelly
,
Marco
Farren-Dai
,
Saeideh
Shamsi Kazem Abadi
,
Verena
Oehler
,
Oluwafemi
Akintola
,
Jason
Draper
,
Michael
Meanwell
,
Saswati
Chakladar
,
Katarzyna
Świderek
,
Vicent
Moliner
,
Robert
Britton
,
Tracey M.
Gloster
,
Andrew J.
Bennet
Diamond Proposal Number(s):
[10071, 14980]
Open Access
Abstract: Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate’s structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.
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Aug 2018
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