Sónia S. Leal

The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron centre

The SOR (sulphur oxygenase reductase) is the initial enzyme in the sulphur-oxidation pathway of Acidianus ambivalens. Expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferric ions were present in the refolding solution. Wild-type, recombinant and refolded SOR possessed indistinguishable properties. Conformational stability studies showed that the apparent unfolding free energy in water is approx. 5 kcal x mol(-1) (1 kcal=4.184 kJ), at pH 7. The analysis of the quaternary structures showed a ball-shaped assembly with a central hollow core probably consisting of 24 subunits in a 432 symmetry. The subunits form homodimers as the building blocks of the holoenzyme. Iron was found in the wild-type enzyme at a stoichiometry of one iron atom/subunit. EPR spectroscopy of the colourless SOR resulted in a single isotropic signal at g=4.3, characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulphur at elevated temperature. Thus both EPR and chemical analysis indicate the presence of a mononuclear iron centre, which has a reduction potential of -268 mV at pH 6.5. Protein database inspection identified four SOR protein homologues, but no other significant similarities. The spectroscopic data and the sequence comparison led to the proposal that the Acidianus ambivalens SOR typifies a new type of non-haem iron enzyme containing a mononuclear iron centre co-ordinated by carboxylate and/or histidine ligands.

Structural Requirements for VAP-B Oligomerization and Their Implication in Amyotrophic Lateral Sclerosis-associated VAP-B(P56S) Neurotoxicity

The integral endoplasmic reticulum (ER)-membrane protein VAP-B interacts with various lipid-transfer/binding proteins containing an FFAT motif through its N-terminal MSP domain. A genetic mutation within its MSP domain, P56S, was identified in familial forms of motor neuron diseases. This mutation induces the formation of insoluble VAP-B(P56S) protein aggregates by an unknown mechanism. In this study, we defined the structural requirements for VAP-B oligomerization and demonstrated their contribution for VAP-B(P56S) aggregation and neurotoxicity. We show that the oligomerization of VAP-B is mainly mediated by its coiled-coil domain and that the GXXXG dimerization motif within the transmembrane domain mediates transmembrane domains self-association but is insufficient to drive VAP-B oligomerization. We further show that the oligomerization of the wild-type VAP-B is independent of its MSP domain. However, we found that the P56S mutation induces conformational changes within the MSP domain and facilitates its propensity to aggregate by exposing hydrophobic patches to the solvent. These conformational changes have no direct effect on FFAT binding. Rather, they enhance VAP-B(P56S) oligomerization driven by the combined contributions of the coiled-coil and the transmembrane domains, thereby preventing accessibility to FFAT-binding site, facilitating the production of VAP-B(P56S)-insoluble aggregates and consequently its neurotoxicity. These results shed light on the mechanism by which VAP-B(P56S) aggregates are formed and induce familial motor neuron diseases.

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid: A LINK TO TOXIC EFFECTS OF CALCIUM OVERLOAD IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)?*

Background: SOD1-enriched protein inclusions and Ca2+ overload are hallmarks in ALS-affected motor neurons. Ca2+ burden correlates with SOD1 aggregation in cellular models. Results: Ca2+ induces conformational changes that enhance and shift SOD1 aggregation from fibrils toward amorphous aggregates. Conclusion: SOD1 aggregation is enhanced and modulated by Ca2+. Significance: Ca2+ can behave as a pathogenic effector in the formation of ALS proteinaceous inclusions. Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca2+ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca2+. We show that at physiological pH, Ca2+ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca2+ boosts the onset of SOD1 aggregation. In agreement, Ca2+ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca2+ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca2+-induced aggregates, thus indicating that Ca2+ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca2+ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca2+ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.

Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron-sulfur center assembly

The biological insertion of iron–sulfur clusters (Fe–S) involves the interaction of (metallo) chaperons with a partly folded target polypeptide. In this respect, the study of nonnative protein conformations in iron–sulfur proteins is relevant for the understanding of the folding process and cofactor assembly. We have investigated the formation of a molten globule state in the [3Fe4S][4Fe4S] ferredoxin from the thermophilic archaeon Acidianus ambivalens (AaFd), which also contains a structural zinc site. Biophysical studies have shown that, at acidic pH, AaFd retains structural folding and metal centers. However, upon increasing the temperature, a series of successive modifications occur within the protein structure: Fe–S disassembly, loss of tertiary contacts and dissociation of the Zn2+ site, which is simultaneous to alterations on the secondary structure. Upon cooling, an apo‐ferredoxin state is obtained, with characteristics of a molten globule: compactness identical to the native form; similar secondary structure evidenced by far‐UV CD; no near‐UV CD detected tertiary contacts; and an exposure of the hydrophobic surface evidenced by 1‐anilino naphthalene‐8‐sulfonic acid (ANS) binding. In contrast to the native form, this apo ferredoxin state undergoes reversible thermal and chemical unfolding. Its conformational stability was investigated by guanidinium chloride denaturation and this state is ∼1.5 kcal mol−1 destabilised in respect to the holo ferredoxin. The single tryptophan located nearby the Fe–S pocket probed the conformational dynamics of the molten globule state: fluorescence quenching, red edge emission shift analysis and resonance energy transfer to bound ANS evidenced a restricted mobility and confinement within a hydrophobic environment. The possible physiological relevance of molten globule states in Fe–S proteins and the hypothesis that their structural flexibility may be important to the understanding of metal center insertion are discussed. Proteins 2007.

S100A6 amyloid fibril formation is calcium-modulated and enhances superoxide dismutase-1 (SOD1) aggregation

Background: The calcium and zinc binding S100A6 protein is overexpressed in ALS and Alzheimers disease. Results: S100A6 aggregates into fibrils under physiological conditions, a process repressed by calcium. Native S100A6 enhances aggregation of SOD1, a hallmark of ALS. Conclusion: S100A6 is a novel amyloidogenic protein and its aggregation is modulated by calcium. Significance: S100A6 aggregation elicits yet unconsidered roles in human pathology. S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

Calcium dysregulation links ALS defective proteins and motor neuron selective vulnerability

More than 20 distinct gene loci have so far been implicated in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by progressive neurodegeneration of motor neurons (MN) and death. Most of this distinct set of ALS-related proteins undergoes toxic deposition specifically in MN for reasons which remain unclear. Here we overview a recent body of evidence indicative that mutations in ALS-related proteins can disrupt fundamental Ca2+ signalling pathways in MN, and that Ca2+ itself impacts both directly or indirectly in many ALS critical proteins and cellular processes that result in MN neurodegeneration. We argue that the inherent vulnerability of MN to dysregulation of intracellular Ca2+ is deeply associated with discriminating pathogenicity and aberrant crosstalk of most of the critical proteins involved in ALS. Overall, Ca2+ deregulation in MN is at the cornerstone of different ALS processes and is likely one of the factors contributing to the selective susceptibility of these cells to this particular neurodegenerative disease.

The prominent conformational plasticity of lactoperoxidase: A chemical and pH stability analysis

Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective C(m) values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.

Intrinsically disordered and aggregation prone regions underlie β-aggregation in S100 proteins.

S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether β-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from α-helices to intermolecular β-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that β-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.

Crystallographic analysis of the intact metal centres [3Fe–4S]1+/0 and [4Fe–4S]2+/1+ in a Zn2+-containing ferredoxin

Detailed structural models of di‐cluster seven‐iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors’ role in protein stability. The here reported, hemihedric twinned crystal structure at 2.0 Å resolution from Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N‐terminal extension comprising a His/Asp Zn2+ site and the ferredoxin (βαβ)2 core, which harbours intact clusters I and II, a [3Fe–4S]1+/0 and a [4Fe–4S]2+/1+ centres. This is in contrast with the previously available ferredoxin structure from Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two [3Fe–4S]1+/0 centres and poor definition around cluster II.

Linear three-iron centres are unlikely cluster degradation intermediates during unfolding of iron-sulfur proteins.

Abstract Recent studies on the chemical alkaline degradation of ferredoxins have contributed to the hypothesis that linear three-iron centres are commonly observed as degradation intermediates of iron-sulfur clusters. In this work we assess the validity of this hypothesis. We studied different proteins containing iron-sulfur clusters, iron-sulfur centres and di-iron centres with respect to their chemical degradation kinetics at high pH, in the presence and absence of exogenous sulfide, to investigate the possible formation of linear three-iron centres during protein unfolding. Our spectroscopic and kinetic data show that in these different proteins visible absorption bands at 530 and 620 nm are formed that are identical to those suggested to arise from linear three-iron centres. Iron release and protein unfolding kinetics show that these bands result from the formation of iron sulfides at pH 10, produced by the degradation of the iron centres, and not from rearrangements leading to linear three-iron centres. Thus, at this point any relevant functional role of linear three-iron centres as cluster degradation intermediates in iron-sulfur proteins remains elusive.