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Abstract: Objective: To determine the high-resolution structure of human Copper-Zinc superoxide dismutase (hSOD1), an antioxidant enzyme whose mutations cause amyotrophic lateral sclerosis (ALS), under near-physiological conditions. Because SOD1 is intrinsically dynamic, capturing its structure at ambient temperature is key to understanding how temperature modulates its conformational flexibility, ensemble and functional states relevant to both catalysis and disease. Materials and Methods: Recombinant hSOD1 was expressed in E. coli, purified by affinity and size-exclusion chromatography, and crystallized at ambient temperature. Serial synchrotron crystallography (SSX) data were collected at 293 K at the EMBL P14-2 Time-Resolved Experiments with Crystallography (T-REXX) beamline at PETRA III, and compared with a 100 K cryogenic at the Diamond Light Source beamline (I03). Both datasets were processed and refined using CCP4 suite and PHENIX packages. B-factor distributions, per-residue RMSD values, and conformational differences were analyzed to quantify temperature-dependent effects. Results: The ambient-temperature SOD1SSX structure was determined at 2.3 Å resolution (PDB ID:9XJ0 this work) and closely matched its 2.37 Å cryogenic counterpart (SOD1CRYO, PDB ID:9XJI this work), both obtained from identical crystallization conditions in the hexagonal P6₃ space group. Cryocooling caused a 3.8% contraction in unit-cell volume, consistent with lattice densification and a 5.2% reduction in molecular surface volume. Despite the overall similarities, the ambient-temperature model revealed localized conformational differences in solvent-exposed loop residues, particularly Ser25-Asn26, Leu67-Glu77, Ile99, and the Asp109-His110-Cys111 triad, and a distinct side-chain orientation of Asn53 was observed at the dimerization interface. While the β-barrel core remained rigid, these regions correspond to redox- and metal-responsive sites implicated in aggregation/fiber formation and putative drug binding. Conclusions: Temperature perturbs local dynamics in SOD1 structure without altering its native dimeric form. The ambient-temperature model reveals flexible, chemically accessible regions that act as druggable hotspots and coincide with ALS-linked mutation sites driving misfolding and aggregation. Considering temperature effects is crucial for structure-based drug design, ensuring candidate molecules engage physiologically relevant conformations. This structure lays the groundwork for future time-resolved crystallography of SOD1 folding and ligand interactions.

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Abstract: Frataxin is a 23 kDa mitochondrial iron-binding protein involved in the biogenesis of iron–sulfur (Fe–S) clusters. Deficiency in frataxin is associated with Friedreich's ataxia, a progressive neurodegenerative disorder. CyaY, the bacterial ortholog of eukaryotic frataxin, is believed to function as an iron donor in Fe–S cluster assembly, making it a key target for structural and functional studies. In this work, a comprehensive structural analysis of the Escherichia coli CyaY protein is presented, comparing its structure at room temperature and cryogenic conditions. Notably, the first room-temperature structures are obtained using the Turkish Light Source “Turkish DeLight” X-ray diffractometer and serial synchrotron X-ray crystallography, marking a significant step forward in understanding CyaY under near-physiological conditions.

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Abstract: Mutations in the E3 ubiquitin ligase Parkin gene have been linked to early onset Parkinson’s disease. Besides many other roles, Parkin is involved in clearance of damaged mitochondria via mitophagy—a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognizes its substrates for ubiquitination. Here, we characterize a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results may explain fast kinetics of Miro1 ubiquitination by Parkin in recombinant assays and provide a biochemical explanation for Miro1-dependent Parkin recruitment to the mitochondrial membrane observed in cells. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson’s disease.

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Abstract: The presence of α-synuclein (α-syn) aggregates, such as Lewy bodies in patients with Parkinson’s disease (PD), contributes to dopaminergic cell death. Injection of PD patient–derived α-syn in nonhuman primates has illustrated the exquisite vulnerability of primate dopaminergic neurons. Here, we aimed to elucidate the temporal and spatial pathological changes induced by two distinct α-syn pathogenic structures, having large or small sizes. To unravel the underlying molecular pathways, we conducted a proteomic analysis of the putamen and the entorhinal cortex, two brain regions carrying notable α-syn pathology. We demonstrate that distinct assemblies of α-syn aggregates drive unique pathogenic changes that ultimately result in a comparable extent of nigrostriatal degeneration at the level of nigral dopaminergic neuron cell bodies and striatal dopaminergic terminals. More broadly, our findings identify pathogenic trajectories associated with large or small α-syn aggregates, suggesting the existence of several possible concomitant pathogenic routes in PD.

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Abstract: Superoxide dismutase 1 (SOD1) is a crucial enzyme that protects cells from oxidative damage by converting superoxide radicals into H2O2 and O2. This detoxification process, essential for cellular homeostasis, relies on a precisely orchestrated catalytic mechanism involving the copper cation, while the zinc cation contributes to the structural integrity of the enzyme. This study presents the 2.3 Å crystal structure of human SOD1 (PDB ID: 9IYK), revealing an assembly of six homodimers and twelve distinct active sites. The water molecules form a complex hydrogen-bonding network that drives proton transfer and sustains active site dynamics. Our structure also uncovers subtle conformational changes that highlight the intrinsic flexibility of SOD1, which is essential for its function. Additionally, we observe how these dynamic structural features may be linked to pathological mutations associated with amyotrophic lateral sclerosis (ALS). By advancing our understanding of hSOD1’s mechanistic intricacies and the influence of water coordination, this study offers valuable insights for developing therapeutic strategies targeting ALS. Our structure’s unique conformations and active site interactions illuminate new facets of hSOD1 function, underscoring the critical role of structural dynamics in enzyme catalysis. Moreover, we conducted a molecular docking analysis using SOD1 for potential radical scavengers and Abelson non-receptor tyrosine kinase (c-Abl, Abl1) inhibitors targeting misfolded SOD1 aggregation along with oxidative stress and apoptosis, respectively. The results showed that CHEMBL1075867, a free radical scavenger derivative, showed the most promising docking results and interactions at the binding site of hSOD1, highlighting its promising role for further studies against SOD1-mediated ALS.