Versatile Fluorescence Lifetime-Based Copper Probe to Quantify Mitochondrial Membrane Potential and Reveal Its Interaction with Protein Aggregation.

Mitochondria play a crucial role in maintaining cellular homeostasis, and the depolarization of mitochondrial membrane potential (MMP) is an important signal of apoptosis. Additionally, protein misfolding and aggregation are closely related to diseases including neurodegenerative diseases, diabetes, and cancers. However, the interaction between MMP changes and disease-related protein aggregation was rarely studied. Herein, we report a novel "turn-on" fluorescent probe MitoRhB that specifically targets to mitochondria for Cu2+ detection in situ. The fluorescence lifetime (τ) of MitoRhB exhibits a positive correlation with MMP changes, allowing us to quantitatively determine the relative MMP during SOD1 (A4 V) protein aggregation. Finally, we found that (1) the increasing concentrations of copper will accelerate the depolarization of mitochondria and reduce MMP; (2) the depolarization of mitochondria can intensify the degree of protein aggregation, suggesting a new routine of copper-induced cell death mediated through abnormal MMP depolarization and protein aggregation.

Early Aggregation Mechanism of SOD128-38 Based on Force Field Parameter of 5-Cyano-Tryptophan.

The aggregation of superoxide dismutase 1 (SOD1) results in amyloid deposition and is involved in familial amyotrophic lateral sclerosis, a fatal motor neuron disease. There have been extensive studies of its aggregation mechanism. Noncanonical amino acid 5-cyano-tryptophan (5-CN-Trp), which has been incorporated into the amyloid segments of SOD1 as infrared probes to increase the structural sensitivity of IR spectroscopy, is found to accelerate the overall aggregation rate and potentially modulate the aggregation process. Despite these observations, the underlying mechanism remains elusive. Here, we optimized the force field parameters of 5-CN-Trp and then used molecular dynamics simulation along with the Markov state model on the SOD128-38 dimer to explore the kinetics of key intermediates in the presence and absence of 5-CN-Trp. Our findings indicate a significantly increased probability of protein aggregate formation in 5CN-Trp-modified ensembles compared to wildtype. Dimeric ß-sheets of different natures were observed exclusively in the 5CN-Trp-modified peptides, contrasting with wildtype simulations. Free-energy calculations and detailed analyses of the dimer structure revealed augmented interstrand interactions attributed to 5-CN-Trp, which contributed more to peptide affinity than any other residues. These results explored the key events critical for the early nucleation of amyloid-prone proteins and also shed light on the practice of using noncanonical derivatives to study the aggregation mechanism.

Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent Kd ~ µM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins.

In silico analysis of SOD1 aggregation inhibition modes of tertiary amine pyrazolone and pyrano coumarin ferulate as ALS drug candidates.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, the familial form (fALS) of which is often cognate to mutations in the antioxidant enzyme Cu/Zn superoxide dismutase 1 (SOD1) leading to misfolding and aggregation. Two small molecules, a tertiary amine pyrazolone (TAP) and a pyrano coumarin ferulate (PCF) were suggested to be ALS drug candidates following experimental observation of their ability to inhibit SOD1 protein misfolding and aggregation. The present work aims at computational investigation of these experimentally proposed drug candidates to gain insight into their mechanism of SOD1 misfolding and aggregation inhibition. On the basis of molecular docking, molecular dynamics simulation, MM-PBSA and per-residue energy decomposition analysis, we examined the specific interactions of TAP and PCF with three probable binding sites of SOD1, namely, dimeric interface cavity, W32 and, UMP binding sites. Results suggest that the binding of TAP at W32 and at UMP sites are least probable due to absence of any favorable interaction. The binding of TAP to dimeric cavity is also unstable due to strong unfavorable interactions. In case of PCF, binding at the UMP site is least probable while binding at dimeric cavity is accompanied by unfavorable interactions. PCF, however, exhibits stable binding with the W32 binding site of SOD1 by stabilizing the solvent accessible hydrophobic residues, which otherwise would have acted as contact points for aggregation. Thus the results imply that compound PCF functions as an inhibitior of SOD1 misfolding/aggregation through direct interaction with the protein SOD1 at the W32 binding site. However, TAP is likely to act as an inhibitor through a different mechanism rather than direct interaction with the protein SOD1. These results apart from reinforcing previous experimental findings, shed light on the probable mechanism of action of the proposed drug candidates.

Polyphenolic flavonoid compounds act as the inhibitory potential of aggregation process: Implications for the prevention and therapeutics against FALS-associated D101G SOD1 mutant.

Aggregation of proteins is a biological phenomenon caused by misfolded proteins. Human superoxide dismutase (hSOD1) misfolding and aggregation underlie the neurological illness amyotrophic lateral sclerosis (ALS). The most significant contributing factor to ALS is genetic point mutations in SOD1. particularly, D101G mutant is the most harmful because it significantly reduces the life expectancy of patients. Subsequently, the use of natural polyphenolic flavonoids is strongly recommended to reduce the amyloidogenic behavior of protopathic proteins. In this study, using computational parameters such as protein-ligand interaction and molecular dynamics (MD) simulation analyses, we are trying to identify a pharmacodynamically promising flavonoid compound that can effectively inhibit the pathogenic behavior of the D101G mutant. Epigallocatechin-gallate (EGCG), Hesperidin, Isorhamnetin, and Diosmetin were identified as potential leads in a preliminary screening of flavonoids to anti-amyloid action. The results of MD showed that the binding of flavonoids to D101G mutant caused changes in stability, hydrophobicity of protein, and flexibility, as well as significantly led to the restoration of lost hydrogen bonds. Secondary structure analysis showed that protein destabilization and the increased propensity of ß-sheet caused by the mutation were restored to the wild-type state upon binding of flavonoids. Besides, to differentiate aggregation, we elucidated alterations in the free energy landscape (FEL) and dynamic cross-correlation matrix (DCCM) of WT-SOD1 and mutant (unbound /bound) states. Among flavonoids, Epigallocatechin-gallate and Hesperidin had the most therapeutic efficacy against the D101G mutant. Therefore, Epigallocatechin-gallate and Hesperidin promise considerable therapeutic potential to develop highly effective inhibitors in reducing fatal and irreversible ALS.

Intracellular spatially-targeted chemical chaperones increase native state stability of mutant SOD1 barrel.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder with currently no cure. Central to the cellular dysfunction associated with this fatal proteinopathy is the accumulation of unfolded/misfolded superoxide dismutase 1 (SOD1) in various subcellular locations. The molecular mechanism driving the formation of SOD1 aggregates is not fully understood but numerous studies suggest that aberrant aggregation escalates with folding instability of mutant apoSOD1. Recent advances on combining organelle-targeting therapies with the anti-aggregation capacity of chemical chaperones have successfully reduce the subcellular load of misfolded/aggregated SOD1 as well as their downstream anomalous cellular processes at low concentrations (micromolar range). Nevertheless, if such local aggregate reduction directly correlates with increased folding stability remains to be explored. To fill this gap, we synthesized and tested here the effect of 9 ER-, mitochondria- and lysosome-targeted chemical chaperones on the folding stability of truncated monomeric SOD1 (SOD1bar) mutants directed to those organelles. We found that compound ER-15 specifically increased the native state stability of ER-SOD1bar-A4V, while scaffold compound FDA-approved 4-phenylbutyric acid (PBA) decreased it. Furthermore, our results suggested that ER15 mechanism of action is distinct from that of PBA, opening new therapeutic perspectives of this novel chemical chaperone on ALS treatment.

Characterization of a novel cysteine-less Cu/Zn-superoxide dismutase in Paenibacillus lautus missing a conserved disulfide bond.

Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.

Structure-based discovery of an antipsychotic drug, paliperidone, as a modulator of human superoxide dismutase 1: a potential therapeutic target in amyotrophic lateral sclerosis.

Aggregates of the antioxidant superoxide dismutase 1 (SOD1) are one of the major contributors to the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in SOD1 lead to an unstable structure and aggregation that perturbs the balance of reactive oxygen species in cells. Oxidation damage to the solvent-exposed Trp32 also causes aggregation of SOD1. Here, the FDA-approved antipsychotic drug paliperidone is identified to interact with Trp32 of SOD1 by structure-based pharmacophore mapping and crystallographic studies. Paliperidone is used for the treatment of schizophrenia. The crystal structure of the complex with SOD1, refined to 2.1 Šresolution, revealed that the ligand binds to the SOD1 ß-barrel in the ß-strand 2 and 3 regions, which are known to scaffold SOD1 fibrillation. The drug also makes substantial π-π interaction with Trp32. Microscale thermophoresis studies confirm significant binding affinity of the compound, suggesting that the ligand can inhibit or prevent tryptophan oxidation. Thus, the antipsychotic drug paliperidone or a derivative may avert SOD1 aggregation and can be used as a lead for ALS drug development.

A computational strategy for therapeutic development against superoxide dismutase (SOD1) amyloid formation: effect of polyphenols on the various events in the aggregation pathway.

Pathology of superoxide dismutase 1 (SOD1) aggregation is linked to a neurodegenerative disease known as amyotrophic lateral sclerosis (ALS). Without suitable post-translational modifications (PTMs), the protein structure tends to become aggregation-prone. Understanding the role of PTMs and targeting the aggregation-prone SOD1 with small molecules can be used to design a strategy to inhibit its aggregation. Microsecond long molecular dynamics (MD) simulations followed by free energy surface (FES) analyses show that the loss of structure in the apo monomer happens locally and stepwise. Removing the disulfide bond from apoprotein leads to further instability in the zinc-binding loop, giving rise to non-native protein conformations. Further, it was found that these non-native conformations have a higher propensity to form a non-native dimer. We chose three structurally similar polyphenols based on their binding energies and investigated their impact on SOD1 aggregation kinetics. MD simulations of apo-SOD1SH/corkscrew fibril-polyphenol complexes were also carried out. The effect of polyphenols was seen on fibril elongation as well. Based on the experiments and MD simulation results, it can be inferred that the choice of inhibitors is influenced not only by the binding energy but also by dimer interface stabilization, the proclivity to form non-native dimers, the propensity to break fibrils, and the propensity to decrease the rate of elongation. The polyphenols with 3' and 4' hydroxyl groups are better inhibitors of SOD1 aggregation.

A liquid-to-solid phase transition of Cu/Zn superoxide dismutase 1 initiated by oxidation and disease mutation.

Cu/Zn superoxide dismutase 1 (SOD1) has a high propensity to misfold and form abnormal aggregates when it is subjected to oxidative stress or carries mutations associated with amyotrophic lateral sclerosis. However, the transition from functional soluble SOD1 protein to aggregated SOD1 protein is not completely clear. Here, we propose that liquid-liquid phase separation (LLPS) represents a biophysical process that converts soluble SOD1 into aggregated SOD1. We determined that SOD1 undergoes LLPS in vitro and cells under oxidative stress. Abnormal oxidation of SOD1 induces maturation of droplets formed by LLPS, eventually leading to protein aggregation and fibrosis, and involves residues Cys111 and Trp32. Additionally, we found that pathological mutations in SOD1 associated with ALS alter the morphology and material state of the droplets and promote the transformation of SOD1 to solid-like oligomers which are toxic to nerve cells. Furthermore, the fibrous aggregates formed by both pathways have a concentration-dependent toxicity effect on nerve cells. Thus, these combined results strongly indicate that LLPS may play a major role in pathological SOD1 aggregation, contributing to pathogenesis in ALS.

A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation.

The misfolding and mutation of Cu/Zn superoxide dismutase (SOD1) is commonly associated with amyotrophic lateral sclerosis (ALS). SOD1 can accumulate within stress granules (SGs), a type of membraneless organelle, which is believed to form via liquid-liquid phase separation (LLPS). Using wild-type, metal-deficient, and different ALS disease mutants of SOD1 and computer simulations, we report here that the absence of Zn leads to structural disorder within two loop regions of SOD1, triggering SOD1 LLPS and amyloid formation. The addition of exogenous Zn to either metal-free SOD1 or to the severe ALS mutation I113T leads to the stabilization of the loops and impairs SOD1 LLPS and aggregation. Moreover, partial Zn-mediated inhibition of LLPS was observed for another severe ALS mutant, G85R, which shows perturbed Zn-binding. By contrast, the ALS mutant G37R, which shows reduced Cu-binding, does not undergo LLPS. In addition, SOD1 condensates induced by Zn-depletion exhibit greater cellular toxicity than aggregates formed by prolonged incubation under aggregating conditions. Overall, our work establishes a role for Zn-dependent modulation of SOD1 conformation and LLPS properties that may contribute to amyloid formation.

Amyotrophic lateral sclerosis disease-related mutations disrupt the dimerization of superoxide dismutase 1 - A comparative molecular dynamics simulation study.

More than 150 genes are involved in amyotrophic lateral sclerosis (ALS), with superoxide dismutase 1 (SOD1) being one of the most studied. Mutations in SOD1 gene, which encodes the enzyme SOD1 is the second most prevalent and studied cause of familial ALS. SOD1 is a ubiquitous, homodimeric metalloenzyme that forms a critical component of the cellular defense against reactive oxygen species. Several mutations in the SOD1 enzyme cause misfolding, dimerization instability, and increased aggregate formation in ALS. However, there is a lack of information on the dimerization of SOD1 monomers and the mechanistic underpinnings on how the pathogenic mutations disrupt the dimerization mechanism. Here, we presented microsecond-scale molecular dynamics (MD) simulations to unravel how interface-based mutations compromise SOD1 dimerization and provide mechanistic understanding into the corresponding process using WT and three interface-based mutant systems (A4V, T54R, and I113T). Structural stability analysis showed that the mutant systems displayed disparate variations in the catalytic sites which may directly alter the stability and activity of the SOD1 enzyme. Based on the dynamic network analysis and principal component analysis, it has been identified that the mutations weakened the correlated motions along the dimer interface and altered the protein conformational behavior, thus weakening the stability of dimer formation. Moreover, the simulation results identified crucial residues such as G51, D52, G114, I151, and Q153 in establishing the dimerization interaction network, which were weakened or absent in the presence of interfacial mutants. Surface potential analysis on mutant systems also displayed changes in the dimerization potential, thus showing the unfavorable dimer formation. Furthermore, network analysis identified the hotspot residues necessary for SOD1 signal transduction which were surprisingly found in the catalytic sites rather than the anticipated dimerization interface.

Structural analysis of the overoxidized Cu/Zn-superoxide dismutase in ROS-induced ALS filament formation.

Eukaryotic Cu, Zn-superoxide dismutase (SOD1) is primarily responsible for cytotoxic filament formation in amyotrophic lateral sclerosis (ALS) neurons. Two cysteine residues in SOD1 form an intramolecular disulfide bond. This study aims to explore the molecular mechanism of SOD1 filament formation by cysteine overoxidation in sporadic ALS (sALS). In this study, we determined the crystal structure of the double mutant (C57D/C146D) SOD1 that mimics the overoxidation of the disulfide-forming cysteine residues. The structure revealed the open and relaxed conformation of loop IV containing the mutated Asp57. The double mutant SOD1 produced more contagious filaments than wild-type protein, promoting filament formation of the wild-type SOD1 proteins. Importantly, we further found that HOCl treatment to the wild-type SOD1 proteins facilitated their filament formation. We propose a feasible mechanism for SOD1 filament formation in ALS from the wild-type SOD1, suggesting that overoxidized SOD1 is a triggering factor of sALS. Our findings extend our understanding of other neurodegenerative disorders associated with ROS stresses at the molecular level.

Metal migration and subunit swapping in ALS-linked SOD1: Zn2+ transfer between mutant and wild-type occurs faster than the rate of heterodimerization.

The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔGHet) between WT and ALS-mutant SOD1 in mismatched metalation states-where one subunit is metalated and the other is not-have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by "stealing" metal ions remains untested. This study used capillary zone electrophoresis and mass spectrometry to track heterodimerization and metal transfer between WT SOD1, ALS-variant SOD1 (E100K, E100G, D90A), and triply deamidated SOD1 (modeled with N26D/N131D/N139D substitutions). We determined that rates of subunit exchange between apo dimers and metalated dimers-expressed as time to reach 30% heterodimer-ranged from t30% = 67.75 ± 9.08 to 338.53 ± 26.95 min; free energies of heterodimerization ranged from ΔGHet = -1.21 ± 0.31 to -3.06 ± 0.12 kJ/mol. Rates and ΔGHet values of partially metalated heterodimers were more similar to those of fully metalated heterodimers than apo heterodimers, and largely independent of which subunit (mutant or WT) was metal-replete or metal-free. Mass spectrometry and capillary electrophoresis demonstrated that mutant or WT 4Zn-SOD1 could transfer up to two equivalents of Zn2+ to mutant or WT apo-SOD1 (at rates faster than the rate of heterodimerization). This result suggests that zinc-replete SOD1 can function as a chaperone to deliver Zn2+ to apo-SOD1, and that WT apo-SOD1 might increase the toxicity of mutant SOD1 by stealing its Zn2+.

Superoxide Dismutase 1 Folding Stability as a Target for Molecular Tweezers in SOD1-Related Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation are hallmarks of many severe neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease. As a supramolecular ligand that binds to lysine and arginine residues, the molecular tweezer CLR01 was found to modify the aggregation pathway of disease-relevant proteins in vitro and in vivo with beneficial effects on toxicity. However, the molecular mechanisms of how tweezers exert these effects remain mainly unknown, hampering further drug development. Here, we investigate the modulation mechanism of unfolding and aggregation pathways of SOD1, which are involved in amyotrophic lateral sclerosis (ALS), by CLR01. Using a truncated version of the wildtype SOD1 protein, SOD1bar , we show that CLR01 acts on the first step of the aggregation pathway, the unfolding of the SOD1 monomer. CLR01 increases, by ∼10 °C, the melting temperatures of the A4V and G41D SOD1 mutants, which are commonly observed mutations in familial ALS. Molecular dynamics simulations and binding free energy calculations as well as native mass spectrometry and mutational studies allowed us to identify K61 and K92 as binding sites for the tweezers to mediate the stability increase. The data suggest that the modulation of SOD1 conformational stability is a promising target for future developments of supramolecular ligands against neurodegenerative diseases.

Effects of Protein Crowders and Charge on the Folding of Superoxide Dismutase 1 Variants: A Computational Study.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) is associated with the misfolding and aggregation of the metalloenzyme protein superoxide dismutase 1 (SOD1) via mutations that destabilize the monomer-dimer interface. In a cellular environment, crowding and electrostatic screening play essential roles in the folding and aggregation of the SOD1 monomers. Despite numerous studies on the effects of mutations on SOD1 folding, a clear understanding of the interplay between crowding, folding, and aggregation in vivo remains lacking. Using a structure-based minimal model for molecular dynamics simulations, we investigate the role of self-crowding and charge on the folding stability of SOD1 and the G41D mutant where experimentalists were intrigued by an alteration of the folding mechanism by a single point mutation from glycine to charged aspartic acid. We show that unfolded SOD1 configurations are significantly affected by charge and crowding, a finding that would be extremely costly to achieve with all-atom simulations, while the native state is not significantly altered. The mutation at residue 41 alters the interactions between proteins in the unfolded states instead of those within a protein. This paper suggests electrostatics may play an important role in the folding pathway of SOD1 and modifying the charge via mutation and ion concentration may change the dominant interactions between proteins, with potential impacts for aggregation of the mutants. This work provides a plausible reason for the alteration of the unfolded states to address why the mutant G41D causes the changes to the folding mechanism of SOD1 that have intrigued experimentalists.

Misfolding-Associated Exposure of Natively Buried Residues in Mutant SOD1 Facilitates Binding to TRAF6.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily impacting motor neurons. Mutations in superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS. Several of these mutations lead to misfolding or toxic gain of function in the SOD1 protein. Recently, we reported that misfolded SOD1 interacts with TNF receptor-associated factor 6 (TRAF6) in the SOD1G93A rat model of ALS. Further, we showed in cultured cells that several mutant SOD1 proteins, but not wildtype SOD1 protein, interact with TRAF6 via the MATH domain. Here, we sought to uncover the structural details of this interaction through molecular dynamics (MD) simulations of a dimeric model system, coarse grained using the AWSEM force field. We used direct MD simulations to identify buried residues, and predict binding poses by clustering frames from the trajectories. Metadynamics simulations were also used to deduce preferred binding regions on the protein surfaces from the potential of the mean force in orientation space. Well-folded SOD1 was found to bind TRAF6 via co-option of its native homodimer interface. However, if loops IV and VII of SOD1 were disordered, as typically occurs in the absence of stabilizing Zn2+ ion binding, these disordered loops now participated in novel interactions with TRAF6. On TRAF6, multiple interaction hot-spots were distributed around the equatorial region of the MATH domain beta barrel. Expression of TRAF6 variants with mutations in this region in cultured cells demonstrated that TRAF6T475 facilitates interaction with different SOD1 mutants. These findings contribute to our understanding of the disease mechanism and uncover potential targets for the development of therapeutics.

Cryo-EM structure of an amyloid fibril formed by full-length human SOD1 reveals its conformational conversion.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. Misfolded Cu, Zn-superoxide dismutase (SOD1) has been linked to both familial and sporadic ALS. SOD1 fibrils formed in vitro share toxic properties with ALS inclusions. Here we produced cytotoxic amyloid fibrils from full-length apo human SOD1 under reducing conditions and determined the atomic structure using cryo-EM. The SOD1 fibril consists of a single protofilament with a left-handed helix. The fibril core exhibits a serpentine fold comprising N-terminal segment (residues 3-55) and C-terminal segment (residues 86-153) with an intrinsic disordered segment. The two segments are zipped up by three salt bridge pairs. By comparison with the structure of apo SOD1 dimer, we propose that eight ß-strands (to form a ß-barrel) and one α-helix in the subunit of apo SOD1 convert into thirteen ß-strands stabilized by five hydrophobic cavities in the SOD1 fibril. Our data provide insights into how SOD1 converts between structurally and functionally distinct states.

Modeling of mutant superoxide dismutase 1 octamers with cross-linked disulfide bonds.

Mutant superoxide dismutase 1 (SOD1) may form cyclic structures due to its greater instability from aberrant demetallization and oxidation of cysteine bonds. This cyclic structure may allow SOD1 to form ion channels on membranes such as the mitochondrial membrane, causing imbalances in the concentration of intracellular ions as a potential mechanism for the progressive neuron death involved in amyotrophic lateral sclerosis (ALS). Using docking programs within modeling software, models of mutant SOD1 dimers and eventually ring oligomers were constructed based on known descriptions of such structures in addition to information on the orientation of the models associated with a membrane. The resulting structure consists of a ring of four demetallated mutant SOD1 dimers with cross-linked disulfide bonds. Stability of the octamer model was supported by the molecular dynamics simulations. Further analysis of the octamer model indicated that its inner- and outer-pore diameters were stable, matching the dimensions of known SOD1 ion channels.

Screening apo-SOD1 conformation stabilizers from natural flavanones using native ion mobility mass spectrometry and fluorescence spectroscopy methods.

RATIONALE: A large number of studies have shown that the production of aberrant and deleterious copper zinc superoxide dismutase (SOD1) species is closely related to amyotrophic lateral sclerosis (ALS). Therefore, it is of great significance to screen effective inhibitors of misfolding and aggregation of SOD1 for treating ALS disease. METHODS: The interaction between flavanone compounds with apo-SOD1was investigated using native electrospray ion mobility mass spectrometry (native ESI-IM-MS). Binding affinities of ligands were compared using native MS, ESI-MS/MS, collision-induced unfolding, and competitive experiments. The effect of ligands on apo-SOD1 aggregation was investigated using the fluorescence spectroscopy method. RESULTS: The results of MS showed that the binding affinity of liquiritin apioside was the strongest, better than the corresponding monosaccharide and aglycone, indicating that the presence and the number of glycosyl group are beneficial to enhance ligand affinity to protein. The results of fluorescence spectroscopy for inhibiting protein aggregation in vitro were consistent with the binding affinity. In addition, the results of the collision-induced unfolding indicated that liquiritin apioside can slow down the unfolding of the protein. Meanwhile, the results of competition experiment suggested that liquiritin apiosides share different binding sites with naringin and 5-fluorouridine, which are significant for the structural stability of SOD1. CONCLUSIONS: This study revealed that the binding of liquiritin apioside can stabilize apo-SOD1 dimer and inhibit the aggregation of apo-SOD1, and illustrated that native ESI-IM-MS is a powerful tool for providing insight into investigating the structure-activity relationship between small molecules and protein, and screening protein conformation stabilizers.