I04-Macromolecular Crystallography
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Rachel L.
Palte
,
Mihir
Mandal
,
Justyna
Sikorska
,
Artjohn B.
Villafania
,
Meredith M.
Rickard
,
Robert J.
Bauer
,
Alexei V.
Buevich
,
Xiaomei
Chai
,
Jiafang
He
,
Zahid
Hussain
,
Markus
Koglin
,
Hannah B.
Macdonald
,
My S.
Mansueto
,
Klaus
Maskos
,
Joey L.
Methot
,
Jaclyn
Robustelli
,
Aileen
Soriano
,
Marcel J.
Tauchert
,
Sriram
Tyagarajan
,
Minjia
Zhang
,
Daniel J.
Klein
,
Jacqueline D.
Hicks
,
David G.
Mclaren
,
Sandra B.
Gabelli
,
Daniel F.
Wyss
Diamond Proposal Number(s):
[35460]
Open Access
Abstract: WRN helicase is an established synthetic lethal target for inhibition in the treatment of microsatellite instability-high (MSI-H) and mismatch repair deficient (MMRd) cancers. The identification of helicase inhibitors is challenging as high-throughput biochemical screening campaigns typically return few validated hits that are often inactive in cell-based assays. Herein, we highlight the power of non-covalent fragment-based lead discovery in locating new druggable allosteric sites on WRN, enabling us to bypass the challenging behavior of WRN during high-throughput screening hampering hit identification. During the fragment optimization process, structures of WRN with key prioritized fragments reveal multiple conformations of WRN with significant domain rotations up to 180°, including a WRN conformation not previously described. Rooted in a combination of biochemical, biophysical, and structural approaches, we present the detailed analyses of optimized chemical matter evolved from screening hits and the unique ability of WRN to accommodate diverse conformations as detailed by structural characterization.
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Jan 2026
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I24-Microfocus Macromolecular Crystallography
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Lídia Dos Passos
Lima
,
Dev
Sriranganadane
,
Daiane Laise
Da Silva
,
Natália C.
Drebes Dörr
,
Enzo Breviglieri Sichi
Mello
,
Caio Vinicius
Dos Reis
,
Rogério Ferreira
Lourenço
,
José Felipe Teixeira Da Silva
Santos
,
Anita
Salmazo
,
Brenno Wendler
Miranda
,
Katlin B.
Massirer
,
Rafael M.
Counago
,
Cristina E.
Alvarez-Martinez
Open Access
Abstract: Serine/threonine (Ser/Thr) kinases of the Hanks-type family are widespread in bacteria, playing key roles in signal transduction. The transmembrane Ser/Thr kinase PknS (XAC4127) from the phytopathogenic bacterium Xanthomonas citri is required for the expression of a type VI secretion system, which confers resistance to predation by the soil amoeba Dictyostelium discoideum. PknS exerts its function via activation of the cognate ECF-type alternative sigma factor EcfK, ultimately triggering the expression of type VI secretion system (T6SS) genes. In this study, we characterize PknS, demonstrating its ability to undergo autophosphorylation both in vitro and within X. citri cells. Structural analysis of the PknS kinase domain revealed the conservation of the canonical fold characteristic of Hanks-type kinases. PknS directly phosphorylates EcfK at five Ser/Thr residues located in two distinct regions of the sigma factor: the conserved σ2 domain (residue T51) and a nonconserved linker connecting domains σ2 and σ4 (residues T104, T106, S108, and S110). The conserved residue T51, previously shown to be essential for sigma factor activity in an EcfK homolog, corresponds to a site that directly interacts with the RNA polymerase β′ subunit. Site-directed mutagenesis analyses further revealed that the conserved residue T106 is also critical for EcfK function. Structural studies indicated that, in addition to T51, phosphorylation at T106 activates EcfK by promoting its interaction with a positively charged pocket within the RNA polymerase β′ subunit. Collectively, our findings describe a previously unknown signal transduction pathway involving a Hanks-type kinase and a sigma factor, providing new insights into the mechanisms of sigma factor activation via phosphorylation in bacteria.
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Jan 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38313]
Open Access
Abstract: Radical enzymes, including glycyl radical enzymes (GREs) and B12-dependent enzymes, catalyze a wide range of biochemical transformations through radical-based mechanisms. An unusual property—conditional resistance to chymotrypsin digestion—has previously been reported for two GREs. However, whether this feature is broadly conserved among related radical enzymes and what factors trigger it has remained unclear. In this study, we investigated five radical enzymes: four GREs and one B12-dependent diol dehydratase. Proteolytic assays demonstrated that substrate binding significantly enhances resistance to chymotrypsin degradation, suggesting a conserved conformational shift from an open, protease-sensitive state to a closed, protease-resistant form. X-ray crystallographic analysis of a GRE-type 1,2-propanediol dehydratase from Raoultella planticola confirmed that active site occupancy correlates with increased protease resistance. Importantly, non-substrate analogs such as 1,3-propanediol and β-methylcholine failed to induce protection, underscoring the specificity of ligand-induced stabilization. These findings reveal a broadly conserved mechanism of substrate-induced conformational stabilization in GREs and B12-dependent radical enzymes and offer a scalable strategy for ligand identification with potential applications in enzyme engineering.
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Dec 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18566]
Open Access
Abstract: Zinc finger antiviral protein (ZAP) is a cytoplasmic protein central to host innate immunity to viral infection. ZAP has no intrinsic catalytic activity but inhibits viral replication by binding to CpG dinucleotides in cytoplasmic viral RNA and recruiting other factors to inhibit protein synthesis and target the RNA for degradation. KHNYN is a ZAP-binding protein required for ZAP-restriction of CpG-rich viral genomes. It contains an extended diKH, PIN nuclease, and CUElike domain, each of which are required for ZAP restriction of viral replication. Here, we report a structural, enzymological, and virological study of KHNYN’s essential PIN nuclease domain. Our crystal structure reveals an extended PIN domain (ex-PIN) containing a conserved N-terminal arm region required for domain stability and an active site tetra-Asp motif, which are both required for antiviral activity. Unlike the weak activity recently reported for the PIN domain, we demonstrate that the KHNYN ex-PIN domain is a highly active Mn2+-dependent single-stranded RNA endonuclease that cleaves with a preference for ApC, ApA, and UpA dinucleotides. These observations extend our view of KHNYN antiviral activity and suggest an unforeseen role for activation by manganese ions in the ZAP–KHNYN antiviral response.
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Dec 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23316]
Abstract: KdpFABC is an ATP-dependent membrane complex that enables prokaryotes to maintain potassium homeostasis under potassium-limited conditions. It features a unique hybrid mechanism combining a channel-like selectivity filter in KdpA with the ATP-driven transport functionality of KdpB. A key unresolved question is whether K+ ions translocate through the inter-subunit tunnel as a queue of ions or individually within a hydrated environment. Using molecular dynamics simulations, metadynamics, anomalous X-ray scattering, and biochemical assays, we demonstrate that the tunnel is predominantly occupied by water molecules rather than multiple K+ ions. Our results identify only one stable intermediate binding site for K+ within the tunnel, apart from the canonical sites in KdpA and KdpB. Free energy calculations reveal a substantial barrier (∼22 kcal/mol) at the KdpA–KdpB interface, making spontaneous K+ translocation unlikely. Furthermore, mutagenesis and functional assays confirm previous findings that Phe232 at this interface plays a key role in coupling ATP hydrolysis to K+ transport. These findings challenge previous models containing a continuous wire of K+ ions through the tunnel and suggest the existence of an as-yet unidentified intermediate state or mechanistic detail that facilitates K+ movement into KdpB.
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Dec 2025
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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):
[38313]
Open Access
Abstract: Mimicry of protein secondary structure elements, such as α-helices and β-sheets, using conformationally constrained peptide macrocycles, can be utilized to disrupt native protein–protein and protein-nucleic acid interactions. Although α-helical stapled peptides have been extensively studied as pharmacological probes, the application of β-sheet and β-hairpin mimetics remains comparatively limited. Less is known about the structural and biophysical consequences of β-hairpin macrocyclization in the context of target binding. In this work, we use a poxvirus immune antagonist protein 018 as a template for the structure-based design of β-hairpin mimetic macrocyclic peptides targeting the STAT1 transcription factor. We demonstrate that successive orthogonal cyclizations have additive effects on the thermodynamic and kinetic properties of peptide binding, most notably slowing the dissociation from the target. We elucidate the structural and dynamic consequences of interstrand and head-to-tail cross-linking and propose a kinetic model explaining the gains in target residence. Finally, we highlight the pharmacological potential of these peptides by competitive inhibition of STAT1 binding to its cognate interferon receptor docking site. These data suggest that β-hairpin macrocyclization may represent a general strategy to extend target engagement, with implications for peptidic probe design.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26835]
Abstract: Pyruvate kinase (PK) is a crucial glycolytic protein involved in vital cellular processes ranging from cell proliferation to immune responses. The activity and functions of PK are tightly regulated by diverse mechanisms, including posttranslational Nϵ-lysine acetylation. Although previous studies have explored the impact of acetylation on selected lysine residues within the M2 isoform of PK (PKM2), a more comprehensive selection of acetylation sites and their respective effects on both PKM2 and the highly homologous PKM1 isoform is lacking. Here, we describe the structural, functional, and regulatory effects of site-specific acetylation on an expanded set of conserved lysines in PKM2 and selected lysines in PKM1. To study homogeneously acetylated proteins, we genetically encoded the incorporation of acetylated lysine into PKM variants expressed in bacteria and cultured mammalian cells. Our integrated biochemical, structural, and computational approach revealed K115 acetylation as an inhibitory modification in both PKM1 and PKM2 that stabilizes a closed active site conformation of the proteins. We also show that, in contrast to K115 acetylation, previously reported acetylation of K305 inhibits PKM2 but has no effect on the activity and oligomerization of PKM1. These findings propose the existence of both uniform and isoform-specific regulatory mechanisms of PKM, mediated by acetylation.
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Dec 2025
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I04-Macromolecular Crystallography
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Thembaninkosi
Gaule
,
Katie J.
Simmons
,
Kieran
Walker
,
Francesco
Del Galdo
,
Rebecca L.
Ross
,
Hema
Viswambharan
,
Jahnavi
Krishnappa
,
Jack
Pacey
,
Martin
Mcphillie
,
Darren C.
Tomlinson
,
Azhar
Maqbool
Open Access
Abstract: Tissue fibrosis is a hallmark of systemic sclerosis (SSc) and results from the persistent activation of fibroblasts and excessive accumulation of extracellular matrix component such as collagen. Recent evidence implicates the matricellular protein Tenascin-C (TNC) in promoting self-sustaining fibroblast activation and fibrosis via its interaction with Toll-like receptor 4 (TLR4). In this study, we utilized Adhiron-guided ligand discovery to identify small molecule inhibitors targeting the fibrinogen-like globe domain of TNC, a key mediator of TLR4 activation. Two lead compounds (464 and 830) demonstrated structural similarity, favourable ADME profiles, and robust anti-fibrotic activity in vitro. Treatment of dermal fibroblasts derived from SSc patients with either compound significantly reduced Transforming growth factor-β-induced expression of fibrotic genes, ACTA2, COL1A1, COL1A2, and CCN2, and inhibited myofibroblast differentiation. These studies may facilitate the development of effective targeted therapy for fibrosis in SSc and support this novel strategy for small molecule development.
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Dec 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[30602]
Open Access
Abstract: Fragment-based drug design offers multiple routes to advance from fragments. One approach is to build structure-activity relationships (SAR) from analogue series in direct-to-biology workflows. Analogues can be prepared by automated chemistry and tested as crude reaction mixtures (CRMs) without purification, but assay noise often leads to hit resynthesis, potentially discarding false negatives and reducing SAR dataset size. High-throughput (HT) X-ray crystallography has the potential to address these issues by resolving hits directly from 100s–1000s of CRMs. However, no systematic analytics exist for extracting SAR models from HT crystallographic evaluation of CRMs. Here, we demonstrate that crystallographic SAR (xSAR) can be extracted from CRMs evaluated via HT X-ray crystallography. We developed a simple rule-based ligand scoring scheme that identifies conserved chemical features associated with crystallographic binding and non-binding. Applied to a crystallographic dataset of 957 fragment elaborations in CRMs targeting PHIP(2), a therapeutically relevant bromodomain, our xSAR model demonstrated effectiveness in two proof-of-concept experiments. First, it recovered 26 missed binders in the initial dataset (false negatives), doubling the hit rate and denoising the dataset. Second, it enabled a prospective virtual screen that identified novel hits with informative chemistries and measurable binding affinities. This work establishes a proof-of-concept that xSAR models can be directly extracted from large-scale crystallographic readouts of CRMs, offering a valuable methodology to build SAR models and accelerate design-make-test iterations without requiring CRM hit resynthesis and confirmation. This invites future work to utilise advanced analytics and modelling techniques to further strengthen purification-agnostic workflows.
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Dec 2025
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