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20 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Structural plasticity of Plasmodium falciparum plasmepsin X to accommodate binding of potent macrocyclic hydroxyethylamine inhibitors Chrislaine Withers-Martinez , Roger George , Roksana Ogrodowicz , Simone Kunzelmann , Andrew G. Purkiss , Svend Kjaer , Philip A. Walker , Vadims Kovada , Aigars Jirgensons , Michael J. Blackman Journal Of Molecular Biology 16 DOI: 10.1016/j.jmb.2025.169062 Open Access Show Abstract ▼ Abstract: Plasmodium falciparum plasmepsin X (PMX) has become a target of choice for the development of new antimalarial drugs due to its essential role across the parasite life cycle. Here we describe the 1.7Å crystallographic structure of PMX noncovalently bound to a potent macrocyclic peptidomimetic inhibitor (7k) possessing a hydroxyethylamine (HEA) scaffold. Upon 7k binding, the enzyme adopts a novel conformation, with significant involvement of the S2’S2 loop (M526-H536) and the S2 flap (F311-G314). This results in partial closure of the active site with widespread interactions in both the prime (S’) and the non-prime (S) sites of PMX. The catalytic aspartate residues D266 and D467 directly interact with the HEA pharmacophore. Docking of a 7k derivative, compound 7a, highlights a region in the S3 pocket near the S3 flexible loop (H242-F248) that may be key for ligand stabilisation. The dynamic nature of PMX and its propensity to undergo distinct types of induced fit upon inhibitor binding enables generation of potent inhibitors that target this essential malarial aspartic protease.	Mar 2025
I04-Macromolecular Crystallography	Structural and functional characterization of the extended-diKH domain from the antiviral endoribonuclease KHNYN Rebecca L. Youle , María José Lista , Clement Bouton , Simone Kunzelmann , Harry Wilson , Matthew A. Cottee , Andrew G. Purkiss , Elizabeth R. Morris , Stuart J. D. Neil , Ian A. Taylor , Chad M. Swanson Journal Of Biological Chemistry 297 DOI: 10.1016/j.jbc.2025.108336 Diamond Proposal Number(s): [25587] Open Access Show Abstract ▼ Abstract: Zinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAP-mediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a non-canonical arrangement between two type-1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor.	Feb 2025
I04-1-Macromolecular Crystallography (fixed wavelength)	Architecture and regulation of a GDNF-GFRα1 synaptic adhesion assembly F. M. Houghton , S. E. Adams , A. S. Ríos , L. Masino , A. G. Purkiss , D. C. Briggs , F. Ledda , N. Q. Mcdonald Nature Communications 14 DOI: 10.1038/s41467-023-43148-8 Diamond Proposal Number(s): [13775] Open Access Show Abstract ▼ Abstract: Glial-cell line derived neurotrophic factor (GDNF) bound to its co-receptor GFRα1 stimulates the RET receptor tyrosine kinase, promoting neuronal survival and neuroprotection. The GDNF-GFRα1 complex also supports synaptic cell adhesion independently of RET. Here, we describe the structure of a decameric GDNF-GFRα1 assembly determined by crystallography and electron microscopy, revealing two GFRα1 pentamers bridged by five GDNF dimers. We reconsitituted the assembly between adhering liposomes and used cryo-electron tomography to visualize how the complex fulfils its membrane adhesion function. The GFRα1:GFRα1 pentameric interface was further validated both in vitro by native PAGE and in cellulo by cell-clustering and dendritic spine assays. Finally, we provide biochemical and cell-based evidence that RET and heparan sulfate cooperate to prevent assembly of the adhesion complex by competing for the adhesion interface. Our results provide a mechanistic framework to understand GDNF-drive	Nov 2023
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 Neil J. Rzechorzek , Simone Kunzelmann , Andrew G. Purkiss , Mariana Silva Dos Santos , James I. Macrae , Ian A. Taylor , Kasper Fugger , Stephen C. West Nature Communications 14 DOI: 10.1038/s41467-023-42544-4 Diamond Proposal Number(s): [25587] Open Access Show Abstract ▼ Abstract: Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery.	Oct 2023
I04-Macromolecular Crystallography	Probing the catalytic mechanism and inhibition of SAMHD1 using the differential properties of Rp- and Sp-dNTPαS diastereomers Elizabeth R. Morris , Simone Kunzelmann , Sarah J. Caswell , Andrew G. Purkiss , Geoff Kelly , Ian A. Taylor Biochemistry DOI: 10.1021/acs.biochem.0c00944 Diamond Proposal Number(s): [13775] Open Access Show Abstract ▼ Abstract: SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2′-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe–Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme–substrate complex.	May 2021
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing Sarah E. Adams , Andrew G. Purkiss , Phillip P. Knowles , Andrea Nans , David C. Briggs , Annabel Borg , Christopher P. Earl , Kerry Goodman , Agata Nawrotek , Aaron J. Borg , Pauline B. Mcintosh , Francesca M. Houghton , Svend Kjær , Neil Mcdonald Structure 15 DOI: 10.1016/j.str.2020.12.012 Diamond Proposal Number(s): [13775] Open Access Show Abstract ▼ Abstract: RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.	Jan 2021
I24-Microfocus Macromolecular Crystallography	Mutations in SKI in Shprintzen–Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization Ilaria Gori , Roger George , Andrew G. Purkiss , Stephanie Strohbuecker , Rebecca A Randall , Roksana Ogrodowicz , Virginie Carmignac , Laurence Faivre , Dhira Joshi , Svend Kjær , Caroline S Hill Elife 10 DOI: 10.7554/eLife.63545 Diamond Proposal Number(s): [13775] Open Access Show Abstract ▼ Abstract: Shprintzen–Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys–Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-β signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.	Jan 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis Elizabeth R. Morris , Sarah J. Caswell , Simone Kunzelmann , Laurence H. Arnold , Andrew G. Purkiss , Geoff Kelly , Ian A. Taylor Nature Communications 11 DOI: 10.1038/s41467-020-16983-2 Diamond Proposal Number(s): [13775, 18566] Open Access Show Abstract ▼ Abstract: SAMHD1 regulates cellular 2′-deoxynucleoside-5′-triphosphate (dNTP) homeostasis by catalysing the hydrolysis of dNTPs into 2′-deoxynucleosides and triphosphate. In CD4+ myeloid lineage and resting T-cells, SAMHD1 blocks HIV-1 and other viral infections by depletion of the dNTP pool to a level that cannot support replication. SAMHD1 mutations are associated with the autoimmune disease Aicardi–Goutières syndrome and hypermutated cancers. Furthermore, SAMHD1 sensitises cancer cells to nucleoside-analogue anti-cancer therapies and is linked with DNA repair and suppression of the interferon response to cytosolic nucleic acids. Nevertheless, despite its requirement in these processes, the fundamental mechanism of SAMHD1-catalysed dNTP hydrolysis remained unknown. Here, we present structural and enzymological data showing that SAMHD1 utilises an active site, bi-metallic iron-magnesium centre that positions a hydroxide nucleophile in-line with the Pα-O5′ bond to catalyse phosphoester bond hydrolysis. This precise molecular mechanism for SAMHD1 catalysis, reveals how SAMHD1 down-regulates cellular dNTP and modulates the efficacy of nucleoside-based anti-cancer and anti-viral therapies.	Jun 2020
I04-1-Macromolecular Crystallography (fixed wavelength)	d-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition Cesira De Chiara , Miha Homšak , Gareth A. Prosser , Holly L. Douglas , Acely Garza-Garcia , Geoff Kelly , Andrew G. Purkiss , Edward W. Tate , Luiz Pedro S. De Carvalho Nature Chemical Biology 5 DOI: 10.1038/s41589-020-0498-9 Diamond Proposal Number(s): [13775] Show Abstract ▼ Abstract: The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.	Mar 2020
I02-Macromolecular Crystallography	IMP1 KH1 and KH2 domains create a structural platform with unique RNA recognition and re-modelling properties Robert Dagil , Neil J Ball , Roksana Ogrodowicz , Fruzsina Hobor , Andrew G. Purkiss , Geoff Kelly , Stephen R Martin , Ian A. Taylor , Andres Ramos Nucleic Acids Research 97 DOI: 10.1093/nar/gkz136 Diamond Proposal Number(s): [13775] Open Access Show Abstract ▼ Abstract: IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2–RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets.	Mar 2019

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