I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692, 19880]
Open Access
Abstract: The innate immune protein human surfactant protein D (SP-D) recognises pathogens in the lungs via binding to carbohydrate surface structures. SP-D targets gram-negative bacterial lipopolysaccharide via calcium-dependent binding, preferentially to the inner core heptose (HepI). To further investigate this recognition, we have determined the high-resolution crystal structures of a trimeric recombinant fragment of human SP-D complexed with synthetic di- and trisaccharides, HepI-Kdo, HepIII-HepII-HepI, and HepII-HepI phosphorylated at either HepI or HepII, inner core lipopolysaccharide motifs common to many gram-negative bacteria. In contrast to acid-hydrolysed lipopolysaccharide used in several previous studies, these synthetic saccharides allow presentation of both the innermost Kdo in its natural pyranose form and heptose phosphorylation. The structures confirm the flexibility of SP-D to adopt alternative binding modes when the preferred epitope is not available, reveal a preference for recognition of the reducing terminal heptose (HepI) via the glyceryl group, indicate that a single Kdo attached to HepI does not have a significant role in ligand recognition, and provide evidence that heptose phosphorylation is a major determinant of recognition. The disaccharide with HepII O4’ phosphorylation binds via the preferred HepI glyceryl-hydroxyls, while HepI O4’ phosphorylation reveals HepII binding via the pyranose ring O3’ and O4’ hydroxyls, which would not be possible with the usual HepII O3’ link to the outer core. The ability of HepI O4’ phosphorylation to prevent preferred HepI recognition suggests a role for heptose phosphorylation in shielding the bacterial LPS inner core from immune recognition.
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Feb 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459, 31353]
Open Access
Abstract: Traboulsi Syndrome is an autosomal recessive hereditary disease associated with developmental defects, in particular of the ocular system. Single nucleotide polymorphisms affecting the ASPH gene, which encodes for the 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH), are associated with Traboulsi Syndrome. AspH catalyzes hydroxylations of conserved aspartate/asparagine residues in epidermal growth factor-like domain (EGFD) proteins. We report studies on the clinically-observed Traboulsi Syndrome-associated R688Q, R735Q, and R735W AspH variants. The results reveal that pathogenic active site substitutions substantially reduce, though do not ablate, EGFD hydroxylase activity compared to wildtype AspH. They imply that efficient AspH catalyzed EGFD hydroxylation is important during human development. Crystallographic studies reveal conservation of the overall AspH fold, but that the preferred conformations of 2OG in complex with the R735Q and R735W AspH variants differ from that with wildtype AspH. Screening of potential 2OG cosubstrate substitutes reveals certain 2-oxoacids, including naturally present metabolites, manifest enhanced catalytic efficiency of Traboulsi Syndrome-associated AspH variants compared to 2OG. The results thus provide proof-of-principle for a therapeutic strategy involving rescue of impaired activities of pathogenic active site AspH variants by use of 2-oxoacids, or 2-oxoacid precursors, other than 2OG.
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Dec 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440, 37593]
Open Access
Abstract: α-Methylacyl-CoA racemase (AMACR; P504S) is a pivotal enzyme involved in the β-oxidation of branched-chain fatty acids and bile acid intermediates, catalyzing the conversion between (2R)- and (2S)-2-methylacyl-CoA thioester epimers. The AMACR reaction enables downstream catabolism of these thioesters via stereospecific enzymes within the β-oxidation pathway. The AMACR homolog in Mycobacterium tuberculosis (MCR) has emerged as a tractable model for dissecting the mechanistic underpinnings of the racemization reaction and presents a promising therapeutic target given the pathogen’s dependence on lipid metabolism for persistence and virulence. Previously we reported the detailed molecular structure of wild-type MCR and in complex with a diverse set of acyl-CoA substrates. They revealed conserved active site residues that mediate substrate anchoring and epimerization and highlighted distinct molecular interactions that confer selectivity toward 2-methyl-branched substrates. Complementing these results, in this report we present high-resolution structures for 2-arylthiopropanoyl-CoA inhibitors in complex with MCR and a comprehensive set of enzyme inhibition assays to delineate structure-activity relationships and probe competitive binding modes. Our findings underscore the importance of inhibitor side-chain branching and CoA anchoring in modulating enzymatic turnover and inhibition. Together, these data enhance our understanding of the racemization mechanism of MCR and establish a structural foundation for the rational design of selective inhibitors. Targeting MCR could represent a novel future therapeutic strategy for M. tuberculosis based on impairing fatty acid utilization.
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Oct 2025
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I04-Macromolecular Crystallography
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Will
Scott
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Esther
Ivorra-Molla
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Dipayan
Akhuli
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Teresa
Massam-Wu
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Pawel K.
Lysyganicz
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Rylie
Walsh
,
Matthew
Parent
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Jonathan
Cook
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Lijiang
Song
,
Abhishek
Kumar
,
Falk
Schneider
,
Masanori
Mishima
,
Allister
Crow
,
Mohan K.
Balasubramanian
Open Access
Abstract: Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric green fluorescent protein recently monomerised through sequence engineering, addresses this challenge with its high photostability. There is now focus on producing different colour StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to red-shift superfolder GFP upon incorporation into its chromophore via genetic code expansion. Here we apply the same strategy to red-shift StayGold through substitution of Tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows an excitation wavelength maximum of 530 nm and an emission wavelength maximum of 588 nm. Importantly, the monomeric mStayRose retains the favourable photostability in vivo in E. coli and zebrafish embryos. A high-resolution crystal structure of StayRose confirms the modified structure of the amino chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step towards developing red-shifted StayGold derivatives.
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Oct 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25587]
Open Access
Abstract: The SspH/IpaH family of novel E3 ligases (NELs) are found in a number of Gram-negative bacteria and are used to target host enzymes for degradation to support pathogenesis. These E3 enzymes are autoinhibited in the absence of substrate and different models for release of autoinhibition have been suggested. However, many of the molecular details of individual steps during the ubiquitin transfer reaction remain unknown. Here, we present the crystal structure of Salmonella SspH1 and an analysis of the solution properties of SspH1 on its own and in complex with substrate and ubiquitin. Our data show that SspH1 exists in a conformational equilibrium between open and closed states and that substrate binding only modulates the distribution of these states but does not induce major conformational changes. This suggests that additional mechanisms must exist to bring the substrates close to the active site to mediate transfer of ubiquitin from the E3∼Ub conjugate.
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Sep 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[35775]
Open Access
Abstract: Non-muscle myosin 2A (NM2A) is the predominant myosin isoform in non-muscle cells. Together with its paralogues NM2B and NM2C, NM2A enables tension and force generation, driving essential cellular processes such as membrane protrusion and retraction, directed migration, adhesion and cytokinesis. The NM2 isoforms display paralogue-specific mechanochemical characteristics that support their specific cellular functions. Here, we determined the structure of the human NM2A motor domain, addressing a critical gap in understanding myosin family diversification. Based on our experimentally resolved 2.1 Å structure of the NM2A motor domain in its nucleotide-free state, we demonstrate, through integrative modeling of NM2-actin complexes and molecular dynamics simulations, how sequence differences between NM2A and NM2B underpin their functional specialization. Loop2 emerges as a critical determinant of isoform-specific behavior. Comparative analysis of ATP interaction fingerprints across NM2 isoforms reveals a conserved ATP binding mechanism. These findings illuminate an allosteric energy transduction pathway that connects sequence variation to actin-binding dynamics, providing mechanistic insight into isoform-specific cytoskeletal functions.
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Sep 2025
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I24-Microfocus Macromolecular Crystallography
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Tomas
Akerud
,
Claudia
De Fusco
,
Peter
Brandt
,
Fredrik
Bergström
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Patrik
Johansson
,
Margareta
Ek
,
Ulf
Börjesson
,
Anders
Johansson
,
Jakob
Danielsson
,
Martin
Bauer
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Bertrand
Arnaud
,
Marie
Castaldo
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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
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Alicia
Moraleda-Montoya
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Zongjia
Chen
,
Ruwan
Epa
,
David
Starns
,
Matthew
Davy
,
Mikel
Garcia-Alija
,
Arnaud
Basle
,
Mario
Schubert
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Didier
Ndeh
,
Beatriz
Trastoy
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Spencer J.
Williams
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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: 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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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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