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89 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Sequence rules for a long SPOP-binding degron required for protein ubiquitylation Linda Makhlouf , Mukul Mishra , Hannah Makhlouf , Iain Manfield , Luca Busino , Elton Zeqiraj Biochemical Journal DOI: 10.1042/BCJ20253041 Diamond Proposal Number(s): [29074, 35120] Open Access Show Abstract ▼ Abstract: The adaptor protein, Speckle-type BTB/POZ protein (SPOP), recruits substrates to the cullin-3-subclass of E3 ligase for selective protein ubiquitylation. The Myddosome protein, Myeloid differentiation primary response 88 (MyD88), is ubiquitylated by the SPOP-based E3 ligase to negatively regulate immune signaling, however, the sequence rules for SPOP-mediated substrate engagement and degradation are not fully understood. Here, we show that MyD88 interacts with SPOP through a long degron that contains the established SPOP-binding consensus and an N-terminal site that we name the Q-motif. Based on sequence similarity to MyD88, we show that additional substrates, including Steroid receptor coactivator-3 (SRC-3), SET domain-containing protein 2 (SETD2) and Caprin1, engage SPOP in this manner. We show that the Q-motif is a critical determinant of these interactions in mammalian cells and determine X-ray crystal structures that show the molecular basis of SPOP associations with these proteins. These studies reveal a new consensus sequence for substrate-binding to SPOP that is necessary for substrate ubiquitylation, thus expanding the sequence rules required for SPOP-mediated E3 ligase substrate recognition.	Mar 2025
I03-Macromolecular Crystallography	Structure, kinetics, and mechanism of Pseudomonas putida sulfoquinovose dehydrogenase, the first enzyme in the sulfoglycolytic Entner-Doudoroff pathway Laura Burchill , Mahima Sharma , Niccolay Madiedo Soler , Ethan D. Goddard-Borger , Gideon J. Davies , Spencer J. Williams Biochemical Journal 482, 57 - 72 DOI: 10.1042/BCJ20240605 Diamond Proposal Number(s): [32736] Open Access Show Abstract ▼ Abstract: The sulfosugar sulfoquinovose (SQ) is catabolized through the sulfoglycolytic Entner-Doudoroff pathway, beginning with the oxidation of SQ to sulfogluconolactone by SQ dehydrogenase. We present a comprehensive structural and kinetic characterization of Pseudomonas putida SQ dehydrogenase (PpSQDH). PpSQDH is a tetrameric enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily with a strong preference for NAD+ over NADP+. Kinetic analysis revealed a rapid equilibrium ordered mechanism in which the NAD+ cofactor is the first substrate to bind, and NADH is the last product to dissociate. Structural studies revealed a homotetrameric structure in solution and crystals, involving cross-subunit interactions in which the C-terminus residue (Gln260) inserts into the diagonally opposite subunit to form part of the second shell of residues lining the active site. Complexes of PpSQDH with SQ or NAD+ provide insight into the recognition of SQ and together with the kinetic analysis allow the proposal of a catalytic reaction mechanism. Our findings illuminate the mechanism of SQ degradation and the evolution of the SDR superfamily for organosulfonate catabolism.	Jan 2025
I24-Microfocus Macromolecular Crystallography	Biophysical and structural analyses of the interaction between the SHANK1 PDZ domain and an internal SLiM Yue Li , Chi H. Trinh , Amanda Acevedo-Jake , Diana Gimenez , Stuart L Warriner , Andrew J. Wilson Biochemical Journal DOI: 10.1042/BCJ20240126 Diamond Proposal Number(s): [19248] Open Access Show Abstract ▼ Abstract: The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterised, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) – EESTSFQGP – as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM – GKAP – FITC-Ahx-EAQTRL-COOH – the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy (FA). To gain further insight on the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganise to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.	Jun 2024
I04-Macromolecular Crystallography	Diversification in the inositol tris/tetrakisphosphate kinase (ITPK) family: crystal structure and enzymology of the outlier AtITPK4 Hayley Whitfield , Sining He , Yinghong Gu , Colleen Sprigg , Hui-Fen Kuo , Tzyy-Jen Chiou , Andrew M. Riley , Barry V. L. Potter , Andrew M. Hemmings , Charles A. Brearley Biochemical Journal DOI: 10.1042/BCJ20220579 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Å resolution that along with description of the enantiospecificity of the enzyme affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a Km for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologs in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.	Mar 2023
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural and functional characterisation of a stable, broad-specificity multimeric sialidase from the oral pathogen Tannerella forsythia Marianne J. Satur , Paulina A. Urbanowicz , Daniel I. R. Spencer , John Rafferty , Graham P. Stafford Biochemical Journal 479, 1785 - 1806 DOI: 10.1042/BCJ20220244 Diamond Proposal Number(s): [17773, 12788] Open Access Show Abstract ▼ Abstract: Sialidases are glycosyl hydrolase enzymes targeting the glycosidic bond between terminal sialic acids and underlying sugars. The NanH sialidase of Tannerella forsythia, one of the bacteria associated with severe periodontal disease plays a role in virulence. Here, we show that this broad-specificity enzyme (but higher affinity for α2,3 over α2,6 linked sialic acids) digests complex glycans but not those containing Neu5,9Ac. Furthermore, we show it to be a highly stable dimeric enzyme and present a thorough structural analysis of the native enzyme in its apo-form and in complex with a sialic acid analogue/ inhibitor (Oseltamivir). We also use non-catalytic (D237A) variant to characterise molecular interactions while in complex with the natural substrates 3- and 6-siallylactose. This dataset also reveals the NanH carbohydrate-binding module (CBM, CAZy CBM 93) has a novel fold made of antiparallel beta-strands. The catalytic domain structure contains novel features that include a non-prolyl cis-peptide and an uncommon arginine sidechain rotamer (R306) proximal to the active site. Via a mutagenesis programme, we identified key active site residues (D237, R212 and Y518) and probed the effects of mutation of residues in proximity to the glycosidic linkage within 2,3 and 2,6-linked substrates. These data revealed that mutagenesis of R306 and residues S235 and V236 adjacent to the acid–base catalyst D237 influence the linkage specificity preference of this bacterial sialidase, opening up possibilities for enzyme engineering for glycotechology applications and providing key structural information that for in silico design of specific inhibitors of this enzyme for the treatment of periodontitis.	Sep 2022
Krios I-Titan Krios I at Diamond	Cryo-EM structures of the Synechocystis sp. PCC 6803 cytochrome b 6 f complex with and without the regulatory PetP subunit Matthew S Proctor , Lorna A. Malone , David A. Farmer , David J. K. Swainsbury , Frederick R. Hawkings , Federica Pastorelli , Thomas Z. Emrich-Mills , C. Alistair Siebert , C. Neil Hunter , Matthew P. Johnson , Andrew Hitchcock Biochemical Journal DOI: 10.1042/BCJ20220124 Diamond Proposal Number(s): [21004, 21005] Open Access Show Abstract ▼ Abstract: In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ~10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn molecule that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.	Jun 2022
I04-1-Macromolecular Crystallography (fixed wavelength)	Divergent binding mode for a protozoan BRC repeat to RAD51 Teodors Pantelejevs , Marko Hyvonen Biochemical Journal DOI: 10.1042/BCJ20220141 Diamond Proposal Number(s): [18548] Open Access Show Abstract ▼ Abstract: Interaction of BRCA2 through ca. 30 amino acid residue motifs, BRC repeats, with RAD51 is a conserved feature of the double-strand DNA break repair by homologous recombination in eukaryotes. In humans the binding of the eight BRC repeats is defined by two sequence motifs, FxxA and LFDE, interacting with distinct sites on RAD51. Little is known of the interaction of BRC repeats in other species, especially in protozoans, where variable number of BRC repeats are found in BRCA2 proteins. Here we have studied in detail the interactions of the two BRC repeats in Leishmania infantum BRCA2 with RAD51. We show LiBRC1 is a high-affinity repeat and determine the crystal structure of its complex with LiRAD51. Using truncation mutagenesis of the LiBRC1 repeat, we demonstrate that high affinity binding is maintained in the absence of an LFDE-like motif and suggest compensatory structural features. These observations point towards a divergent evolution of BRC repeats, where a common FxxA-binding ancestor evolved additional contacts for affinity maturation and fine-tuning.	May 2022
I03-Macromolecular Crystallography	Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase Zhuoyao Chen , Jinwei Zhang , Adrián R. Murillo-De-Ozores , María Castañeda-Bueno , Francesca D'Amico , Raphael Heilig , Charlotte E. Manning , Fiona J. Sorrell , Vincenzo D'Angiolella , Roman Fischer , Monique P. C. Mulder , Gerardo Gamba , Dario R. Alessi , Alex N. Bullock Biochemical Journal 479, 661 - 675 DOI: 10.1042/BCJ20220019 Diamond Proposal Number(s): [10619] Open Access Show Abstract ▼ Abstract: The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1–4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing four amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.	Mar 2022
B21-High Throughput SAXS	Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimerization and multimeric assembly Yashwanth Ashok , Carlos Vela-Rodríguez , Chun-Song Yang , Heli I. Alanen , Fan Liu , Bryce M Paschal , Lari Lehtio Biochemical Journal DOI: 10.1042/BCJ20210722 Diamond Proposal Number(s): [19951, 21035] Open Access Show Abstract ▼ Abstract: Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we have generated recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1:1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.	Jan 2022
B21-High Throughput SAXS	Conformational trapping of an ABC transporter in polymer lipid nanoparticles Naomi L. Pollock , James Lloyd , Carlotta Montinaro , Megha Rai , Timothy R. Dafforn Biochemical Journal 479, 145 - 159 DOI: 10.1042/BCJ20210312 Diamond Proposal Number(s): [15549] Open Access Show Abstract ▼ Abstract: ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC–SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866–SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866–SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866–SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.	Jan 2022

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