I24-Microfocus Macromolecular Crystallography
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Tomas
Akerud
,
Claudia
De Fusco
,
Peter
Brandt
,
Fredrik
Bergström
,
Patrik
Johansson
,
Margareta
Ek
,
Ulf
Börjesson
,
Anders
Johansson
,
Jakob
Danielsson
,
Martin
Bauer
,
Bertrand
Arnaud
,
Marie
Castaldo
,
Maria
Strömstedt
,
Birgitta
Rosengren
,
Frank
Jansen
,
Linda
Fredlund
Diamond Proposal Number(s):
[20016]
Open Access
Abstract: Nicotinamide N-methyl transferase (NNMT) is involved in the regulation of cellular nicotinamide adenine dinucleotide (NAD) and S-adenosyl-L-methionine (SAM) levels and has been implicated in a range of human diseases. Herein, we show that a class of NNMT inhibitors, analogs of the natural substrate nicotinamide (NAM), is turned over by the enzyme and that the methylated product is a potent inhibitor of the enzyme. The product inhibitor is, however, charged and has modest cellular potency. Utilizing this on-target biotransformation combines the cell permeability of the substrate with the high potency of the product resulting in highly efficient inhibition in vivo. First, we studied the structure-activity-relationship for both substrates and methylated products and solved structures using X-ray crystallography of representative inhibitors. Then we designed a new surface biosensor method to understand the structure-kinetic-relationship for the inhibitors. We were able to quantify the substrate binding kinetics to NNMT-SAM, catalysis rate, and rate of product release from NNMT-SAH in a single experiment. This is to our knowledge the first time an enzyme surface biosensor has been used to study and quantify catalysis in detail. Finally, by monitoring plasma concentrations of turnover inhibitor substrate, product, and the endogenous product, 1-Methyl nicotinamide (1-MNA), in the rat, we show that the turnover inhibitor mechanism of action is relevant in vivo.
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Jun 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Zak
Mciver
,
Alicia
Moraleda-Montoya
,
Zongjia
Chen
,
Ruwan
Epa
,
David
Starns
,
Matthew
Davy
,
Mikel
Garcia-Alija
,
Arnaud
Basle
,
Mario
Schubert
,
Didier
Ndeh
,
Beatriz
Trastoy
,
Spencer J.
Williams
,
Marcelo E.
Guerin
,
Alan
Cartmell
Diamond Proposal Number(s):
[18598, 30305, 21970]
Open Access
Abstract: Rhamnogalacturonan II is one of the most complex plant cell wall carbohydrates and is composed of 13 different sugars and 21 different glycosidic linkages. It is abundant in fruit and indulgence foods, such as chocolate and wine, making it common in the human diet. The human colonic commensal Bacteroides thetaiotaomicron expresses a consortium of 22 enzymes to metabolise rhamnogalacturonan II, some of which exclusively target sugars unique to rhamnogalacturonan II. Several of these enzyme families remain poorly described, and, consequently, our knowledge of rhamnogalacturonan II metabolism is limited. Chief among the poorly understood activities is glycoside hydrolase (GH) family 139, with targets α1,2-2O-methyl L-fucoside linkages, a sugar residue a sugar not found in any other plant cell wall complex glycans. Although the founding enzyme BT0984 was placed in the RG-II degradative pathway, no GH139 structure or catalytic blueprint had been available. We report the crystal structures of BT0984 and a second homologue, and reveal that the family operates with inverting stereochemistry. Using this data we undertook a mutagenic strategy, backed by molecular dynamics, to identify the important substrate binding and catalytic residues, mapping these residues throughout the GH139 family revealing the importance of the O2 methyl interaction of the substrate. We propose a catalytic mechanism that uses a non-canonical Asn as a catalytic base and shares similarity with L-fucosidases/L-galactosidases of family GH95.
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Jun 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440]
Open Access
Abstract: α-Methylacyl-CoA racemase (AMACR; P504S) enzyme plays a vital role in branched-chain fatty acid metabolism by catalysing the conversion of 2-methyl-branched fatty acyl-CoAs into a near 1 to 1 mixture of the (2R)- and (2S)-epimers, enabling further metabolism. α-Methylacyl-CoA racemase from Mycobacterium tuberculosis (MCR) has been explored as a model to understand the AMACR racemization mechanism and as a drug target. Here we present a detailed analysis of a new MCR wild-type crystal structure to provide insights into the MCR racemization mechanism and the molecular features that contribute enzyme activity and selectivity. Specifically, we report a structure of wild-type MCR (in tetragonal space group I422, a new crystal form) along with 12 structures of MCR in complex with branched-chain 2-methylacyl-CoA esters (ibuprofenoyl-CoA, ±-fenoprofenoyl-CoA, S-ketoprofenoyl-CoA, ±-flurbiprofenoyl-CoA, S-naproxenoyl-CoA, S-2-methyldecanoyl-CoA, and isobutanoyl-CoA) and straight-chain acyl-CoA esters (decanoyl-CoA, octanoyl-CoA, hexanoyl-CoA, butanoyl-CoA, acetyl-CoA) in the range of 1.88 to 2.40 Å resolution. These detailed molecular structures enhance our understanding of substrate recognition and coupled with extensive enzyme inhibition assays provide a framework for the rational structure-based drug design of selective and potent MCR inhibitors to combat Mycobacterium tuberculosis in the future.
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May 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
Abstract: Listeria monocytogenes is a ubiquitous, psychrotrophic human pathogen that can cause listeriosis, a serious illness for vulnerable populations. Some foods, such as Hispanic-style fresh cheeses like queso fresco, pose a specific risk because there are no widely accepted or available methods for L. monocytogenes mitigation that are both effective and able to maintain the properties of the products. Listeria-specific bacteriophages encode endolysins that are able to cleave the peptidoglycan layer of L. monocytogenes cells externally, showing promise as a potential solution to this problem. PlyP100, from the GRAS Listeria phage P100, is one such endolysin that can prevent the growth of L. monocytogenes in both lab culture conditions and a miniaturized queso fresco model. In this work, we aimed to understand the structural and functional properties of PlyP100. An AlphaFold prediction suggested the presence of three separate domains (D1, D2, and D3). By solving a crystal structure of D1 and assessing various domain truncations, we present evidence that D1 is responsible for catalytic activity, D3 is sufficient for cell wall binding, and D2 is necessary for full function of the enzyme against live cells. Additionally, we performed point mutations in D1 and compared PlyP100 to proteins with similar structures, including S. pneumoniae LytA and Listeria endolysin Ply511, in order to understand its specific enzymatic mechanism and target strain specificity. These insights into the structure and function of PlyP100 will aid future work aiming to engineer better endolysins as safe food antimicrobials.
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May 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440]
Open Access
Abstract: The prodigious ability of bacteria to catabolize aromatic compounds has sparked considerable efforts to engineer bacteria to valorize lignin, an under-utilized component of biomass. Despite decades of study, key catabolic pathways and enzymes remain poorly characterized. We recently identified the hydroxyphenylethanone (Hpe) pathway, which enables Rhodococcus rhodochrous GD02 and other bacteria to catabolize 4-hydroxyacetophenone (HAP) and acetovanillone (AV), which are generated in the catalytic fractionation of lignin. Catabolism is initiated by a two-component, ATP-dependent dikinase, HpeHI, homologs of which are involved in the catabolism of other aromatic compounds. In biochemical studies, the kinase activity of HpeHI was highest at low ionic strength and low concentrations of Mn2+. HpeHI had highest apparent specificity for HAP and AV (kcat/KM ≥ 250 mM-1 s-1) and had submicromolar KM values for these substrates, consistent with the enzyme acting as a scavenging system. The enzyme also transformed 4-hydroxybenzaldehyde, vanillin, acetosyringone, and phenol. A 1.8 Å crystal structure of HpeI revealed that it is homologous to the ATP-grasp domain of rifampin phosphotransferase (RPH) while an AlphaFold model of HpeH indicated that it is homologous to the swivel and rifampin-binding domains of RPH. Consistent with HpeHI using a similar mechanism where the swivel domain transits between the spatially distinct substrate-binding sites, substitution of the conserved His residue in HpeH abolished kinase activity. Moreover, the HpeH component alone catalyzed phosphotransfer from 4-phosphoacetophenone to AV. This study reveals a subfamily of small molecule dikinases that comprise two components, some of which are involved in aromatic compound catabolism.
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May 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[29835]
Open Access
Abstract: We present a comprehensive investigation into the catalytic mechanism of methylisocitrate lyase, a potential drug target candidate against the zoonotic pathogen Coxiella burnetii, the causative agent of Q fever and a federal select agent. Current treatment regimens are prolonged, often with incomplete clearance of the pathogen. We utilised a structure-based bioinformatics pipeline to identify methylisocitrate lyase as a candidate therapeutic target against C. burnetii from a list of essential genes. Wild-type C. burnetii methylisocitrate lyase has a kcat of 13.8 s-1 (compared to 105 s-1 for Salmonella enterica) and isocitrate inhibits with a KI of 11 mM. We have determined the previously uncharacterised substrate-bound structure of this enzyme family, alongside product and inhibitor-bound structures. These structures of wild-type enzyme reveal that in the active state the catalytic C118 is positioned 2.98 Å from O5 of methylisocitrate and Arg152 moves towards the substrate relative to the inhibitor bound structure. Analysis of structure-based mutants reveals that Arg152 and Glu110 are both essential for catalysis. We suggest that Arg152 acts as the catalytic base that initiates the methylisocitrate lyase reaction. These results deepen our understanding of the catalytic mechanism of methylisocitrate lyase and could aid the development of new therapeutics against C. burnetii.
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Apr 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Open Access
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen, commonly associated with human airway infections. Based on its amino acid sequence similarity with Pyrococcus furiosus protease I, P. aeruginosa PfpI was originally annotated as an intracellular protease. In this work, we show that PfpI is a methylglyoxalase. The X-ray crystal structure of the purified protein was solved to 1.4 Å resolution. The structural data indicated that PfpI shares the same constellation of active site residues (including the catalytic Cys112 and His113) as those seen in a well-characterized bacterial methylglyoxalase from Escherichia coli, YhbO. Using NMR, we confirmed that PfpI qualitatively converted methylglyoxal into lactic acid. Quantitation of lactate produced by the methylglyoxalase activity of PfpI yielded a kcat of 102 min-1 and a KM of 369 μM. Mutation of Cys112 and His113 in PfpI led to complete loss of methylglyoxalase activity. To investigate the functional impact of PfpI in vivo, a ΔpfpI deletion mutant was made. Quantitative proteomic analyses revealed a pattern of changes consistent with perturbation of ribosomal function, Zn2+ limitation, C1 metabolism, and glutathione metabolism. These findings are consistent with PfpI being a glutathione-independent methylglyoxalase. Previously, transposon insertion (pfpI::Tn) mutants have been reported to exhibit phenotypes associated with antibiotic resistance, motility and the response to oxidative stress. However, the ΔpfpI mutant generated in this study displayed none of these phenotypes. Whole-genome sequencing of the previously described pfpI::Tn mutants revealed that they also contain a variety of other genetic changes that likely account for their observed phenotypes.
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Mar 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17077, 28224]
Open Access
Abstract: T cell receptors (TCRs) recognize specific peptides presented by human leukocyte antigens (HLAs) on the surface of antigen presenting cells and are involved in fighting pathogens and cancer surveillance. Canonical docking orientation of TCRs to their target peptide-HLAs (pHLAs) is essential for T cell activation, with reverse binding TCRs lacking functionality. TCR binding geometry and molecular interaction footprint with pHLAs is typically obtained by determining the crystal structure. Here, we describe the use of a cross-linking tandem mass spectrometry (XL-MS/MS) method to decipher the binding orientation of several TCRs to their target pHLAs. Cross-linking sites were localized to specific residues and their molecular interactions showed differentiation between TCRs binding in canonical or reverse orientations. Structural prediction and crystal structure determination of two TCR-pHLA complexes validated these findings. The XL-MS/MS method described herein offers a faster and simpler approach for elucidating TCR-pHLA binding orientation and interactions.
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Mar 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18944]
Open Access
Abstract: The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential for regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulate its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pocket predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for the development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits that extensively sample UP1 functional regions involved in RNA recognition and binding, as well as map hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provide rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1-mediated splicing regulation.
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Feb 2025
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[25587]
Open Access
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.
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Feb 2025
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