I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11265]
Open Access
Abstract: Botulinum neurotoxins are the causative agents of botulism, a lethal paralytic disease, but are also one of the most commonly used therapeutics for the treatment of numerous neuromuscular conditions. These toxins recognise motor nerve terminals with high specificity and affinity by using a dual binding mechanism involving gangliosides and protein receptors. The initial recognition of gangliosides is crucial for the toxins’ potency. In this study, we employed a synaptosome-binding screening strategy to identify BoNT/A mutants with enhanced ganglioside-binding which translated into improved potency. X-ray crystallography and receptor-binding assays were used to elucidate the molecular mechanisms underlying the increased affinity or altered ganglioside selectivity of these mutants. Our findings provide a basis for the development of BoNT/A variants with enhanced therapeutic potential.
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Jun 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Human somatic angiotensin I-converting enzyme is a key zinc metallopeptidase in cardiovascular regulation that hydrolyzes angiotensin peptides (Ang I, Ang II), as well as other vasoactive peptides, including kinins (e.g., bradykinin), substance P, the acetylated tetrapeptide Ac-Ser-Asp-Lys-Pro, and the amyloid ß-peptide. Because of its enzymatic promiscuity, ACE and its substrates and products affect many physiological processes, including blood pressure control, hemopoiesis, reproduction, renal development/function, fibrosis, and immune response. ACE inhibitors are among the most important therapeutic agents available today for the treatment of hypertension, heart failure, coronary artery disease, renal insufficiency, and general atherosclerosis. However, they need much improvement because of the side effects seen in patients with long-term treatment due to nonselective inhibition of the N- and C-domains of ACE (referred to as nACE and cACE, respectively). Here, we report that ACE activity can be inhibited by ciprofloxacin, a potent fluoroquinolone antibiotic (IC50 202.7/Ki 33.8 μM for cACE). In addition, the high-resolution crystal structure of cACE in complex with ciprofloxacin reveals that it binds at an exosite away from the active site pocket, overlapping the position of a potential allosteric site with a different binding mode. The detailed structural information reported here will provide a useful scaffold for the design of future potent allosteric inhibitors.
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Jun 2025
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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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VMXi-Versatile Macromolecular Crystallography in situ
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Abstract: Macromolecular X-ray crystallography is a powerful tool enabling modern structure- based drug design, where structures of protein-ligand complexes provide a basis for rational design decisions to improve the potency of ligands. However, like the protein itself, small molecules within protein crystals are subject to specific radiation damage (SRD) during the collection of X-ray diffraction data, but the effect of SRD on small molecule ligands has not yet been extensively described. This study is following on recently published work1 investigating SRD to halogenated ligands in protein-ligand structures of the therapeutic cancer target B-cell lymphoma 6 protein (BCL6), which found significant cleavage of carbon-halogen (C-X) bonds during X-ray diffraction data collection. The present work is making use of an improved set of BCL6 ligands to gain further understanding of the role of the halogen type and substitution position on the susceptibility of the C-X bond to SRD. Additionally, diffraction data will not only be collected at cryogenic temperature, but also at room temperature at the VMXi beamline at Diamond Light Source to investigate the impact of data collection temperature on SRD to small molecule ligands.
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Jun 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26794]
Abstract: ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 μM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 μM. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds.
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Jun 2025
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B21-High Throughput SAXS
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Alexander
Leithner
,
Oskar
Staufer
,
Tanmay
Mitra
,
Falk
Liberta
,
Salvatore
Valvo
,
Mikhail
Kutuzov
,
Hannah
Dada
,
Jacob
Spaeth
,
Weijie
Zhou
,
Felix
Schiele
,
Sophia
Reindl
,
Herbert
Nar
,
Stefan
Hoerer
,
Maureen
Crames
,
Stephen
Comeau
,
David
Young
,
Sarah
Low
,
Edward
Jenkins
,
Simon J.
Davis
,
David
Klenerman
,
Andrew
Nixon
,
Noah
Pefaur
,
David
Wyatt
,
Omer
Dushek
,
Srinath
Kasturirangan
,
Michael L.
Dustin
Open Access
Abstract: Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
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Jun 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Yeojin
Kim
,
Aleksandar
Lučić
,
Christopher
Lenz
,
Frederic
Farges
,
Martin P.
Schwalm
,
Krishna
Saxena
,
Thomas
Hanke
,
Peter G.
Marples
,
Jasmin C.
Aschenbrenner
,
Daren
Fearon
,
Frank
Von Delft
,
Andreas
Kramer
,
Stefan
Knapp
Diamond Proposal Number(s):
[29658]
Open Access
Abstract: Tripartite motif-containing protein 21 (TRIM21), and particularly its PRY-SPRY protein interaction domain, plays a critical role in the immune response by recognizing intracellular antibodies targeting them for degradation. In this study, we performed a crystallographic fragment screening (CFS) campaign to identify potential small molecule binders targeting the PRY-SPRY domain of TRIM21. Our screen identified a total of 109 fragments binding to TRIM21 that were distributed across five distinct binding sites. These fragments have been designed to facilitate straightforward follow-up chemistry, making them ideal starting points for further chemical optimization. A subsequent fragment merging approach demonstrated improved activity. To enable functional validation of compounds with full length human TRIM21, we established a NanoBRET assay suitable for measuring target engagement to the main Fc binding site in life cells. The high-resolution structural data and observed binding modes across the different sites highlight the versatility of the PRY-SPRY domain as a target for small-molecule intervention. The presented data provide a solid foundation for structure-guided ligand design, enabling the rational design of more potent and selective compounds, with the goal to develop bivalent molecules such as Proteolysis Targeting Chimeras (PROTACs).
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Jun 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 28534]
Open Access
Abstract: The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1−GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1−GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1−GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1−GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1−GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1−GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.
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Jun 2025
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NONE-No attached Diamond beamline
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Abstract: Protein–inhibitor crystal structures aid medicinal chemists in efficiently improving the potency and selectivity of small-molecule inhibitors. It is estimated that a quarter of lead molecules in drug dis- covery projects are halogenated. Protein–inhibitor crystal structures have shed light on the role of halogen atoms in ligand binding. They form halogen bonds with protein atoms and improve shape complementarity of inhibitors with protein binding sites. However, specific radiation damage (SRD) can cause cleavage of carbon–halogen (C–X) bonds during X-ray diffraction data collection. This study shows significant C–X bond cleavage in protein–ligand structures of the therapeutic cancer targets B-cell lymphoma 6 (BCL6) and heat shock protein 72 (HSP72) complexed with halogenated ligands, which is dependent on the type of halogen and chemical structure of the ligand. The study found that metrics used to evaluate the fit of the ligand to the electron density deteriorated with in- creasing X-ray dose, and that SRD eliminated the anomalous signal from brominated ligands. A point of diminishing returns is identified, where collecting highly redundant data reduces the anomalous signal that may be used to identify binding sites of low-affinity ligands or for experimental phasing. Straightforward steps are proposed to mitigate the effects of C–X bond cleavage on structures of proteins bound to halogenated ligands and to improve the success of anomalous scattering experiments.
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Jun 2025
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I04-Macromolecular Crystallography
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Jonathan J.
Ford
,
Javier
Santos-Aberturas
,
Edward S.
Hems
,
Joseph W.
Sallmen
,
Lena A. K.
Bögeholz
,
Guy
Polturak
,
Anne
Osbourn
,
Joseph A.
Wright
,
Marina V.
Rodnina
,
Danny
Vereecke
,
Isolde M.
Francis
,
Andrew W.
Truman
Diamond Proposal Number(s):
[32728]
Open Access
Abstract: The natural products actinonin and matlystatin feature an N-hydroxy-2-pentyl-succinamyl (HPS) chemophore that facilitates metal chelation and confers their metalloproteinase inhibitory activity. Actinonin is the most potent natural inhibitor of peptide deformylase (PDF) and exerts antimicrobial and herbicidal bioactivity by disrupting protein synthesis. Here, we used a genomics-led approach to identify candidate biosynthetic gene clusters (BGCs) hypothesized to produce HPS-containing natural products. We show that one of these BGCs is on the pathogenicity megaplasmid of the plant pathogen Rhodococcus fascians and produces lydiamycin A, a macrocyclic pentapeptide. The presence of genes predicted to make an HPS-like chemophore informed the structural recharacterization of lydiamycin via NMR and crystallography to show that it features a rare 2-pentyl-succinyl chemophore. We demonstrate that lydiamycin A inhibits bacterial PDF in vitro and show that a cluster-situated PDF gene confers resistance to lydiamycin A, representing an uncommon self-immunity mechanism associated with the production of a PDF inhibitor. In planta competition assays showed that lydiamycin enhances the fitness of R. fascians during plant colonization. This study highlights how a BGC can inform the structure, biochemical target, and ecological function of a natural product.
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May 2025
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