Aconitate decarboxylase an enzyme member of the MmgE-PrpD family of proteins has gained significant attention in the last decade as a therapeutic target for cancer and inflammatory diseases Its product itaconate is a multifunctional metabolite shown to drive several disease states Though extensively studied in cellulo and in vivo this ...More |Related Solutions: NT8^®

Aconitate decarboxylase 1, an enzyme member of the MmgE-PrpD family of proteins, has gained significant attention in the last decade as a therapeutic target for cancer and inflammatory diseases. Its product, itaconate, is a multifunctional metabolite shown to drive several disease states. Though extensively studied in cellulo and in vivo, this protein is biochemically and mechanistically under characterized and although a family of inhibitors has been described, no ligand-bound structures have yet been determined. In this work we present a thorough structural investigation that yielded the first ligand-bound structure of this protein family, which required the generation of artifact-free apo crystals. We also developed a novel, low-consumption, robust kinetic assay and investigated active site and allosteric mutants to further elucidate structural and dynamic activity relationships of this protein. Less |Related Solutions: NT8^®

Fc RIIa encoded by FCGR A is a widely expressed Fc receptor implicated in autoimmunity and infectious disease susceptibility To fine-map the rheumatoid arthritis RA association at the complex FCGR locus we combined gene-specific resequencing genetic association studies in UK and Spanish European cohorts functional genomics structural biology biophysical analyses ...More |Related Solutions: NT8^®

FcγRIIa, encoded by FCGR2A, is a widely expressed Fc receptor implicated in autoimmunity and infectious disease susceptibility. To fine-map the rheumatoid arthritis (RA) association at the complex FCGR locus, we combined gene-specific resequencing, genetic association studies in UK and Spanish European cohorts, functional genomics, structural biology, biophysical analyses, and cellular assays. We identified a common European FCGR2A haplotype (2A.3), defined by Q27W, H131H, and the RA-associated SNP rs12746613, which showed the strongest association with RA. Multi-omics analyses demonstrated that 2A.3 is associated with reduced expression of the soluble FCGR2A splice variant and lower circulating soluble FcγRIIa levels. Functional studies revealed altered IgG interactions and delayed FcγRIIa signal transduction associated with Q27W, while structural analyses found no evidence for stable ectodomain dimerisation. Together, these findings identify 2A.3 as an important functional contributor to RA susceptibility and provide mechanistic insight into how FCGR2A variation may influence immune regulation and disease risk in Europeans. Less |Related Solutions: NT8^®

The ribosome is a universally conserved and essential protein complex but its biogenesis in mammals is more complex than in single-celled eukaryotes To explore this added complexity we conducted a protein protein interaction screen in human cells This led to the identification of the eumetazoan-specific SPATA SPATA L CINP C ...More |Related Solutions: Rock Imager^®

The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein–protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5–SPATA5L1–CINP–C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5’s ATPase activity is more important than SPATA5L1’s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCC’s role in pre-60S maturation. Less |Related Solutions: Rock Imager^®

Babesia bovis is a species of apicomplexan hemoparasitic protozoa that can be transmitted by ticks causing a global cattle disease As it depends mainly on the glycolytic pathway for energy production and life cycle maintenance glycolytic enzymes are possible targets for drug development against Babesia Glyceraldehyde- -Phosphate Dehydrogenase GAPDH has ...More |Related Solutions: Rock Imager^®

Babesia bovis is a species of apicomplexan hemoparasitic protozoa that can be transmitted by ticks, causing a global cattle disease. As it depends mainly on the glycolytic pathway for energy production and life cycle maintenance, glycolytic enzymes are possible targets for drug development against Babesia. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) has been one of such targets, against several parasitic organisms. It performs the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate in the presence of nicotinamide adenine dinucleotide. The protocol for Babesia bovis Glyceraldehyde-3-Phosphate Dehydrogenase (BbGAPDH) expression and purification has been developed to yields of 28 mg of pure protein per liter of culture medium, with a specific activity of 55.5 ± 6.99 U mg−1 after His-tag removal, indicating preservation of enzymatic activity. For crystallization, the His-tag removal proved essential. Crystals diffracted to 3.12 Å resolution in the P3121 space group; NAD+ cofactor molecules are observed in their respective sites. Comparisons to the theoretical model indicate a number of side chain conformation differences. These results provide support for future enzyme inhibition assays, in addition to crystallization assays with potential inhibitors. Less |Related Solutions: Rock Imager^®

NADP-dependent malic enzyme NADP-ME has been repeatedly co-opted into distinct metabolic roles across plants most prominently as the decarboxylase of NADP-ME-type C photosynthesis In maize the plastidic C - and nonC -NADP-ME isoforms are closely related in sequence yet display strikingly different catalytic properties suggesting that small changes in ligand ...More |Related Solutions: Rock Imager^®

NADP-dependent malic enzyme (NADP-ME) has been repeatedly co-opted into distinct metabolic roles across plants, most prominently as the decarboxylase of NADP-ME-type C4 photosynthesis. In maize, the plastidic C4- and nonC4-NADP-ME isoforms are closely related in sequence yet display strikingly different catalytic properties, suggesting that small changes in ligand recognition can re-tune reaction chemistry. However, mechanistic interpretation has been hampered by the scarcity of plant NADP-ME structures captured in catalytically informative, ligand-bound states. Here, we integrate X-ray crystallography with structure-guided docking and atomistic molecular dynamics (MD) to resolve ligand-site interaction networks across reaction states. We determined a 2.55 Å structure of maize plastidic nonC4-NADP-ME bound to NADP+, pyruvate, and Mg2+, revealing a conserved NADP-ME fold with localized active-site flexibility. Comparison with maize C4-NADP-ME uncovers isoform-specific rewiring of NADP+ and pyruvate contacts, with the nonC4 enzyme forming a denser product–cofactor interaction network. To access substrate-bound states, we reconstructed malate-NADP+-Mg2+ complexes by docking followed by MD, identifying distinct malate-Mg2+ coordination geometries and alternative NADP+ positioning between isoforms. Together, these structures and simulations provide a network-level framework for plastidic NADP-ME functional diversification and generate testable hypotheses for how ligand coordination drives isoform-specific catalysis. Less |Related Solutions: Rock Imager^®