Carlos Alfonso

Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation

Significance Certain oligomeric species generated during the self-assembly of specific proteins into ordered fibrillar aggregates are likely to be key players in the initiation and spreading of neurodegenerative diseases. We have purified stable toxic oligomeric species of α-synuclein and defined and minimized their degree of heterogeneity, which has allowed us to identify distinct subgroups of oligomers and determine their structural properties and three-dimensional molecular architectures. All the oligomeric subgroups possess approximately cylindrical architectures with marked similarities to amyloid fibrils, suggesting that these types of oligomers are kinetically trapped during protein self-assembly. The relative stabilities and inherent pathological roles of different amyloid oligomers are likely to result from the multiplicity of pathways of the misfolding process and the remarkably slow rates of structural conversions. We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.

The GTPase activity of Escherichia coli FtsZ determines the magnitude of the FtsZ polymer bundling by ZapA in vitro

FtsZ polymerizes in a ring-like structure at mid cell to initiate cell division in Escherichia coli. The ring is stabilized by a number of proteins among which the widely conserved ZapA protein. Using antibodies against ZapA, we found surprisingly that the cellular concentration of ZapA is approximately equal to that of FtsZ. This raised the question of how the cell can prevent their interaction and thereby the premature stabilization of FtsZ protofilaments in nondividing cells. Therefore, we studied the FtsZ−ZapA interaction at the physiological pH of 7.5 instead of pH 6.5 (the optimal pH for FtsZ polymerization), under conditions that stimulate protofilament formation (5 mM MgCl2) and under conditions that stimulate and stabilize protofilaments (10 mM MgCl2). Using pelleting, light scattering, and GTPase assays, it was found that stabilization and bundling of FtsZ polymers by ZapA was inversely correlated to the GTPase activity of FtsZ. As GTP hydrolysis is the rate-limiting factor for depolymerization of FtsZ, we propose that ZapA will only enhance the cooperativity of polymer association during the transition from helical filament to mid cell ring and will not stabilize the short single protofilaments in the cytoplasm. All thus far published in vitro data on the interaction between FtsZ and ZapA have been obtained with His-ZapA. We found that in our case the presence of a His tag fused to ZapA prevented the protein to complement a ΔzapA strain in vivo and that it affected the interaction between FtsZ and ZapA in vitro.

Purification of a heat-stable chitin deacetylase from Aspergillus nidulans and its role in cell wall degradation.

An extracellular chitin deacetylase activity has been purified to homogeneity from autolyzed cultures of Aspergillus nidulans. This enzyme is an acidic glycoprotein with a pI of 2.75 and a 28% (wt/wt) carbohydrate content. The apparent Mr of the enzyme estimated by SDS/PAGE and Superose 12 (f.p.l.c.) was around 27,000. The enzyme had an optimum pH at 7.0 and was stable in the pH range 4.0–7.5. Its optimum temperature of reaction was 50°C, and it was stable from 30° to 100°C after 1 h of preincubation. The enzyme hydrolyzed glycol chitin and oligomers of N-acetylgucosamine and to a lesser extent chitin, colloidal chitin, carboxymethylchitin, and an α-1 → 3,1 → 6-N-acetylgalactosamine-galactan among other substances with amido groups, but the enzyme did not hydrolyze peptide bonds. The role of this enzyme could be deacetylation of chitin oligosaccharides during autolysis, after action of endochitinase on cell walls.

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T2RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T2RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T2RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T2RB3, T2Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.

Infectious bursal disease virus is an icosahedral polyploid dsRNA virus

Viruses are a paradigm of the economy of genome resources, reflected in their multiplication strategy and for their own structure. Although there is enormous structural diversity, the viral genome is always enclosed within a proteinaceous coat, and most virus species are haploid; the only exception to this rule are the highly pleomorphic enveloped viruses. We performed an in-depth characterization of infectious bursal disease virus (IBDV), a non-enveloped icosahedral dsRNA virus with a bisegmented genome. Up to 6 natural populations can be purified, which share a similar protein composition but show higher sedimentation coefficients as particle density increases. Stoichiometry analysis of their genome indicated that these biophysical differences correlate with the copy number of dsRNA segments inside the viral capsid. This is a demonstration of a functional polyploid icosahedral dsRNA virus. We show that IBDV particles with greater genome copy number have higher infectivity rates. Our results show an unprecedented replicative strategy for dsRNA viruses and suggest that birnaviruses are living viral entities encompassing numerous functional and structural characteristics of positive and negative ssRNA viruses.

Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates DIFFERENTIAL CHEMOTACTIC RESPONSE TOWARDS RECEPTOR LIGANDS

We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (Tm) and unfolding enthalpy (ΔH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination

CBMs (carbohydrate-binding modules) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. They have been frequently identified by amino acid sequence alignments, but only a few have been experimentally established to have a carbohydrate-binding activity. A small olive pollen protein, Ole e 10 (10 kDa), has been described as a major inducer of type I allergy in humans. In the present study, the ability of Ole e 10 to bind several polysaccharides has been analysed by affinity gel electrophoresis, which demonstrated that the protein bound 1,3-beta-glucans preferentially. Analytical ultracentrifugation studies confirmed binding to laminarin, at a protein/ligand ratio of 1:1. The interaction of Ole e 10 with laminarin induced a conformational change in the protein, as detected by CD and fluorescence analyses, and an increase of 3.6 degrees C in the thermal denaturation temperature of Ole e 10 in the presence of the glycan. These results, and the absence of alignment of the sequence of Ole e 10 with that of any classified CBM, indicate that this pollen protein defines a novel family of CBMs, which we propose to name CBM43. Immunolocalization of Ole e 10 in mature and germinating pollen by transmission electron microscopy and confocal laser scanning microscopy demonstrated the co-localization of Ole e 10 and callose (1,3-beta-glucan) in the growing pollen tube, suggesting a role for this protein in the metabolism of carbohydrates and in pollen tube wall re-formation during germination.

Sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus, is a monomer-tetramer associating protein

Sticholysin II (Stn‐II) is a pore‐forming cytolysin. Stn‐II interacts with several supports for size exclusion chromatography, which results in an abnormal retardation precluding molecular mass calculations. Sedimentation equilibrium analysis has revealed that the protein is an associating system at neutral pH. The obtained data fit a monomer‐tetramer equilibrium with an association constant K c 4 of 109 M−3. The electrophoretic pattern of Stn‐II treated with different cross‐linking reagents, in a wide range of protein concentrations, corroborates the existence of tetrameric forms in solution. A planar configuration of the four monomers, C4 or D2 symmetry, is proposed from modelling of the cross‐linking data.

Effect of aggregation state on the toxicity of different amphotericin B preparations.

The aim of this work was to study the effect of aggregation of amphotericin B (AMB) in their toxicity. The aggregation of AMB depends on different formulation factors such as pH and excipients, therefore three formulations with different AMB aggregation states were prepared: a monomeric form (M-AMB), a dimeric form (D-AMB) and a poly-aggregated form (P-AMB). The predominant aggregation state of each AMB formulation was characterized by spectrophotometry and their size by dynamic laser light scattering. Toxicity was evaluated by lethality in mice and hemolysis test from human erythrocytes and the experimental AMB formulations were compared with reference formulations of AmBisome, Fungizone and heated Fungizone. The less toxic aggregation form of AMB was the poly-aggregated one which was similar to AmBisome. Moreover, the P-AMB and heated Fungizone were centrifuged to isolate different size fractions. The toxicity of these two heterogeneous formulations was related to their size, so the smaller the aggregation size fraction the higher the toxicity determined by hemolysis. It can be concluded that the aggregation state of AMB and their size affects critically the toxicity of AMB. The low toxic P-AMB formulations contain a different poly-aggregated state to that of AmBisome, heated Fungizone and the other studied AMB aggregation states.

Structural Analysis of Tobacco Etch Potyvirus HC-Pro Oligomers Involved in Aphid Transmission

ABSTRACT Oligomeric forms of the HC-Pro protein of the tobacco etch potyvirus (TEV) have been analyzed by analytical ultracentrifugation and single-particle electron microscopy combined with three-dimensional (3D) reconstruction. Highly purified HC-Pro protein was obtained from plants infected with TEV by using a modified version of the virus that incorporates a histidine tag at the HC-Pro N terminus (hisHC-Pro). The purified protein retained a high biological activity in solution when tested for aphid transmission. Sedimentation equilibrium showed that the hisHC-Pro preparations were heterogenous in size. Sedimentation velocity confirmed the previous observation and revealed that the active protein solution contained several sedimenting species compatible with dimers, tetramers, hexamers, and octamers of the protein. Electron microscopy fields of purified protein showed particles of different sizes and shapes. The reconstructed 3D structures suggested that the observed particles could correspond to dimeric, tetrameric, and hexameric forms of the protein. A model of the interactions required for oligomerization of the HC-Pro of potyviruses is proposed.