I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Christian
Roth
,
Olga V.
Moroz
,
Suzan A. D.
Miranda
,
Lucas
Jahn
,
Elena V.
Blagova
,
Andrey A.
Lebedev
,
Dorotea R.
Segura
,
Mary A.
Stringer
,
Esben P.
Friis
,
Joao P. L.
Franco Cairo
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Endo-galactosaminidases are an underexplored family of enzymes involved in the degradation of galactosaminogalactan (GAG) and other galactosamine-containing cationic exopolysaccharides produced by fungi and bacteria. These exopolysaccharides are part of the cell wall and extracellular matrix of microbial communities. Currently, these galactosaminidases are found in three distinct CAZy families: GH114, GH135 and GH166. Despite the widespread occurrence of these enzymes in nearly all bacterial and fungal clades, only limited biochemical and structural data are available for these three groups. To expand our knowledge of endo-galactosaminidases, we selected several sequences predicted to encode endo-galactosaminidases and produced them recombinantly for structural and functional studies. Only very few predicted proteins could be produced in soluble form, and activity against bacterial Pel (pellicle) polysaccharide could only be confirmed for one enzyme. Here, we report the structures of two bacterial and one fungal enzyme. Whereas the fungal enzyme belongs to family GH114, the two bacterial enzymes do not lie in the current GH families but instead define a new family, GH191. During structure solution we realized that crystals of all three enzymes had various defects including twinning and partial disorder, which in the case of a more severe pathology in one of the structures required the design of a specialized refinement/model-building protocol. Comparison of the structures revealed several features that might be responsible for the described activity pattern and substrate specificity compared with other GAG-degrading enzymes.
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May 2025
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[31336]
Open Access
Abstract: Pathogens have evolved diverse strategies to counteract host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates ‘eat me’ signals for antibacterial autophagy, but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here, we show that the enterobacterium Shigella flexneri actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to RNF213, ubiquitylates it and targets it for proteasomal degradation, thus counteracting host-protective LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the cryogenic electron microscopy structure of the IpaH1.4–RNF213 complex. The specificity of the interaction is achieved through the leucine-rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding to ubiquitin-charged E2 enzymes. IpaH1.4 also targets other E3 ligases involved in inflammation and immunity through binding to the E2-interacting face of their RING domains, including the E3 ligase LUBAC that is required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213. We conclude that IpaH1.4 has evolved to antagonize multiple antibacterial and proinflammatory host E3 ligases.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Plasmodium falciparum plasmepsin X (PMX) has become a target of choice for the development of new antimalarial drugs due to its essential role across the parasite life cycle. Here we describe the 1.7Å crystallographic structure of PMX noncovalently bound to a potent macrocyclic peptidomimetic inhibitor (7k) possessing a hydroxyethylamine (HEA) scaffold. Upon 7k binding, the enzyme adopts a novel conformation, with significant involvement of the S2’S2 loop (M526-H536) and the S2 flap (F311-G314). This results in partial closure of the active site with widespread interactions in both the prime (S’) and the non-prime (S) sites of PMX. The catalytic aspartate residues D266 and D467 directly interact with the HEA pharmacophore. Docking of a 7k derivative, compound 7a, highlights a region in the S3 pocket near the S3 flexible loop (H242-F248) that may be key for ligand stabilisation. The dynamic nature of PMX and its propensity to undergo distinct types of induced fit upon inhibitor binding enables generation of potent inhibitors that target this essential malarial aspartic protease.
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Mar 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Virginie
Will
,
Lucile
Moynie
,
Elise
Si Ahmed Charrier
,
Audrey
Le Bas
,
Lauriane
Kuhn
,
Florian
Volck
,
Johana
Chicher
,
Hava
Aksoy
,
Morgan
Madec
,
Cyril
Antheaume
,
Gaëtan L. A.
Mislin
,
Isabelle J.
Schalk
Diamond Proposal Number(s):
[33133]
Abstract: Iron is essential for bacterial growth, and Pseudomonas aeruginosa synthesizes the siderophores pyochelin (PCH) and pyoverdine to acquire it. PCH contains a thiazolidine ring that aids in iron chelation but is prone to hydrolysis, leading to the formation of 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde (IQS). Using mass spectrometry, we demonstrated that PCH undergoes hydrolysis and oxidation in solution, resulting in the formation of aeruginoic acid (AA). This study used proteomic analyses and fluorescent reporters to show that AA, dihydroaeruginoic acid (DHA), and PCH induce the expression of femA, a gene encoding the ferri-mycobactin outer membrane transporter in P. aeruginosa. Notably, the induction by AA and DHA was observed only in strains unable to produce pyoverdine, suggesting their weaker iron-chelating ability compared to that of pyoverdine. 55Fe uptake assays demonstrated that both AA-Fe and DHA-Fe complexes are transported via FemA; however, no uptake was observed for PCH-Fe through this transporter. Structural studies revealed that FemA is able to bind AA2-Fe or DHA2-Fe complexes. Key interactions are conserved between FemA and these two complexes, with specificity primarily driven by one of the two siderophore molecules. Interestingly, although no iron uptake was noted for PCH through FemA, the transporter also binds PCH-Fe in a similar manner. These findings show that under moderate iron deficiency, when only PCH is produced by P. aeruginosa, degradation products AA and DHA enhance iron uptake by inducing femA expression and facilitating iron transport through FemA. This provides new insights into the pathogen’s strategies for iron homeostasis.
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Mar 2025
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Krios V-Titan Krios V at Diamond
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Diamond Proposal Number(s):
[31098, 37220]
Open Access
Abstract: The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) regulates the level of cholesterol by catalysing the formation/production of mevalonate and has therefore become an important pharmaceutical target for coronary heart disease. Here, we report the cryo-EM structure of the catalytic part of the enzyme in the apo form and bound with its inhibitor atorvastatin, a commonly used drug in cardiovascular disease, at resolutions of 2.1 and 2.3 Å, respectively. In the cryo-EM maps, part of the N-domain corresponding to amino acids 439–487 is well ordered and could be modelled completely. Atorvastatin molecules were found to occupy all four active sites of the tetrameric complex, and the binding does not alter the conformation of the protein or the active site. The method described here exploits graphene oxide as an additional support and could be used as an alternative to elucidate the structures of pharmaceutical target compounds that are difficult to co-crystallize with human HMGR and for sparsely available samples in drug discovery.
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Mar 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Alexandra
Males
,
Olga V.
Moroz
,
Elena
Blagova
,
Astrid
Munch
,
Gustav H.
Hansen
,
Annette H.
Johansen
,
Lars H.
Østergaard
,
Dorotea R.
Segura
,
Alexander
Eddenden
,
Anne V.
Due
,
Martin
Gudmand
,
Jesper
Salomon
,
Sebastian R.
Sørensen
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Joao Paulo L.
Franco Cairo
,
Mark
Nitz
,
Roland A.
Pache
,
Rebecca M.
Vejborg
,
Sandeep
Bhosale
,
David J.
Vocadlo
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-D-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering.
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Mar 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269, 21265]
Open Access
Abstract: Mono-ADP-ribosyl transferase (mART) proteins are secreted virulence factors produced by several human pathogens, the founding member being diphtheria toxin (DT). Pseudomonas aeruginosa can also secrete a mART toxin, known as exotoxin A (PE), but with an organization of its three functional domains (receptor, translocation, and enzymatic elements) that is opposite to DT. Two additional PE-like toxins (PLTs) have been identified from Vibrio cholerae and Aeromonas hydrophila, suggesting more PLT family members may exist. Database mining discovered six additional putative homologues, considerably extending this group of PLTs across a wide range of bacterial species. Here, we examine sequence and structural information for these new family members with respect to previously identified PLTs. The X-ray crystal structures of four new homologues show the conservation of critical features responsible for structure and function. This study shows the potential of these newly described toxins for the development of novel drug delivery platforms. Additionally, genomic analysis suggests horizontal gene transfer to account for the wide distribution of PLTs across a range of eubacteria species, highlighting the need to monitor emerging pathogens and their virulence factors.
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Mar 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[40457, 32977, 32479]
Open Access
Abstract: The basic helix–loop–helix PER-ARNT-SIM (bHLH-PAS) proteins BMAL1 and CLOCK heterodimerize to form the master transcription factor governing rhythmic gene expression. Owing to connections between circadian regulation and numerous physiological pathways, targeting the BMAL1–CLOCK complex pharmacologically is an attractive entry point for intervening in circadian-related processes. In this study, we developed a small molecule, Core Circadian Modulator (CCM), that targets the cavity in the PASB domain of BMAL1, causing it to expand, leading to conformational changes in the PASB domain and altering the functions of BMAL1 as a transcription factor. Biochemical, structural and cellular investigations validate the high level of selectivity of CCM in engaging BMAL1, enabling direct access to BMAL1–CLOCK cellular activities. CCM induces dose-dependent alterations in PER2–Luc oscillations and orchestrates the downregulation of inflammatory and phagocytic pathways in macrophages. These findings collectively reveal that the BMAL1 protein architecture is inherently configured to enable the binding of chemical ligands for functional modulation.
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Mar 2025
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I23-Long wavelength MX
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Diamond Proposal Number(s):
[26794]
Abstract: Perlecan is an essential multi-domain, disulfide bond rich basement membrane protein. Mutations in perlecan cause Schwartz-Jampel syndrome and dyssegmental dysplasia. While there has been a large body of experimental work reported on perlecan, there is only minimal structural information available to date. There is no prior structural data for region 3 of perlecan in which some Schwartz-Jampel syndrome causing point mutations have been reported. Here, we produce constructs of the disulfide rich region 3 of perlecan along with five mutations previously reported to cause Schwatz-Jampel syndrome. Four of the mutations resulted in decreased yields and thermal stability compared to the wild-type protein. In contrast, the P1019L mutation was produced in good yields and showed higher thermal stability than the wild-type protein. The crystal structures for both the wild-type and P1019L mutation were solved. As expected, both showed laminin IV-like and laminin-type EGF-like domains, with the P1019L mutation resulting in only a minor conformational change in a loop region and no significant changes in regular secondary or tertiary structure.
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Mar 2025
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I03-Macromolecular Crystallography
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Andry Mercedes
Mavila
,
Jhon Antoni
Vargas
,
Eloy
Condori
,
Erick Giancarlo
Suclupe Farro
,
Adriano
Alves Furtado
,
Josué Manuel
López
,
Silvia Lucila
Gonzalez
,
Humberto D’muniz
Pereira
,
Jorge Luis
Marapara
,
Roger Ruiz
Paredes
,
Marianela
Cobos
,
Juan C.
Castro
,
Richard Charles
Garratt
,
Diego Antonio
Leonardo
Diamond Proposal Number(s):
[31229]
Abstract: Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (Ankistrodesmus sp.). Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg2+-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from Ankistrodesmus sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in Ankistrodesmus sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.
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Mar 2025
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