José M. Mancheño

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.

BzdR, a Repressor That Controls the Anaerobic Catabolism of Benzoate in Azoarcus sp. CIB, Is the First Member of a New Subfamily of Transcriptional Regulators

In this work, we have studied the transcriptional regulation of the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB. The transcription start site of the PN promoter running the expression of the bzd catabolic genes was identified. Gel retardation assays and PN::lacZ translational fusion experiments performed both in Azoarcus sp. CIB and Escherichia coli cells have shown that bzdR encodes a specific repressor that controls the inducible expression of the adjacent bzd catabolic operon, being the first intermediate of the catabolic pathway (i.e. benzoyl-CoA, the actual inducer molecule). This is the first report of a transcriptional repressor and a CoA-derived aromatic inducer controlling gene expression in the anaerobic catabolism of aromatic compounds. DNase I footprinting experiments revealed that BzdR protected three regions (operators) at the PN promoter. The three operators contain direct repetitions of a TGCA sequence that forms part of longer palindromic structures. In agreement with the repressor role of BzdR, operator region I spans the transcription initiation site as well as the -10 sequence for recognition of the RNA polymerase. Primary sequence analyses of BzdR showed an unusual modular organization with an N-terminal region homologous to members of the HTH-XRE family of transcriptional regulators and a C-terminal region similar to shikimate kinases. A three-dimensional model of the N-terminal and C-terminal regions of BzdR, generated by comparison with the crystal structures of the SinR regulator from Bacillus subtilis and the shikimate kinase I protein from E. coli, strongly suggests that they contain the helix-turn-helix DNA-binding motif and the benzoyl-CoA binding groove, respectively. The BzdR protein constitutes, therefore, the prototype of a new subfamily of transcriptional regulators.

Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin

α‐Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis—histidine‐50, glutamic acid‐96, and histidine‐137—were changed to glutamine. Three dif‐ ferent single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three‐dimensional structure of these variants of α‐sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of α‐sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild‐type α‐sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α‐sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α‐sarcin. Proteins 1999;37:474–484. ©1999 Wiley‐Liss, Inc.

The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease

The hydrolysis of adenylyl(3′→5′)adenosine (ApA) and guanylyl(3′→5′)adenosine (GpA) dinucleotides by the cytotoxic protein α‐sarcin has been studied. Quantitative analysis of the reaction has been performed through reverse‐phase chromatographic (HPLC) separation of the resulting products. The hydrolysis of the 3′‐5′ phosphodiester bond of these substrates yields the 2′‐3′ cyclic mononucleotide; this intermediate is converted into the corresponding 3′‐monophosphate derivative as the final product of the reaction. The values of the apparent Michaelis constant (K M), k cat and k cat/K M have also been calculated. The obtained results fit into a two‐step mechanism for the enzymatic activity of α‐sarcin and allow to consider this protein as a cyclizing RNase.

Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus

Two major proteins, alpha-sarcin and an antifungal polypeptide (AFP), are secreted by the mould Aspergillus giganteus MDH 18894 when it is cultured for 70-80 h. A third major protein is also found in the extracellular medium at 48-60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH2-terminal primary structure, amino acid content, spectroscopical features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP (lf-AFP). Its amino acid composition is identical to that of AFP but containing six extra residues. NH2-terminal sequence analysis of the first eight amino acid residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be Ile/Leu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue (Asp/Glu) and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.

Two-Dimensional Crystallization on Lipid Monolayers and Three-Dimensional Structure of Sticholysin II, a Cytolysin from the Sea Anemone Stichodactyla helianthus

Sticholysin II (Stn II), a potent cytolytic protein isolated from the sea anemone Stichodactyla helianthus, has been crystallized on lipid monolayers. With Fourier-based methods, a three-dimensional (3D) model of Stn II, up to a resolution of 15 A, has been determined. The two-sided plane group is p22(1)2, with dimensions a = 98 A, b = 196 A. The 3D model of Stn II displays a Y-shaped structure, slightly flattened, with a small curvature along its longest dimension (51 A). This protein, with a molecular mass of 19. 2 kDa, is one of the smallest structures reconstructed with this methodology. Two-dimensional (2D) crystals of Stn II on phosphatidylcholine monolayers present a unit cell with two tetrameric motifs, with the monomers in two different orientations: one with its longest dimension lying on the crystal plane and the other with this same axis leaning at an angle of approximately 60 degrees with the crystal plane.

Partially folded states of the cytolytic protein sticholysin II.

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.

Involvement of the amino-terminal β-hairpin of the Aspergillus ribotoxins on the interaction with membrances and nonspecific ribonuclease activity

Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three‐dimensional structures of two of these proteins, α‐sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino‐terminal β‐hairpin of α‐sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of α‐sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid‐perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of α‐sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid‐interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino‐terminal β‐hairpin in the interaction with both membranes and polyadenylic acid.

OLIGOMERIZATION OF THE CYTOTOXIN ALPHA -SARCIN ASSOCIATED WITH PHOSPHOLIPID MEMBRANES

alpha-Sarcin is a cytotoxic protein that specifically inactivates ribosomes. The protein translocates across phospholipid membranes. Oligomerization of the protein occurs upon interaction with membranes. Chemically cross-linked protein oligomers have been obtained by treatment of protein-vesicle complexes with the membrane impermeant reagent bis-(sulfosuccinimidyl) suberate. These structures are only obtained in the presence of acidic lipid vesicles composed of either natural or synthetic phospholipids. Such oligomers are not produced in concentrated protein solutions in the absence of vesicles. The formation of the chemically stabilized oligomers is saturated at the same lipid to protein molar ratio as all the perturbations caused by alpha-sarcin on lipid vesicles. Results are discussed in terms of the involvement of oligomer formation on protein translocation across membranes.

Identification of a Missing Link in the Evolution of an Enzyme into a Transcriptional Regulator

The evolution of transcriptional regulators through the recruitment of DNA-binding domains by enzymes is a widely held notion. However, few experimental approaches have directly addressed this hypothesis. Here we report the reconstruction of a plausible pathway for the evolution of an enzyme into a transcriptional regulator. The BzdR protein is the prototype of a subfamily of prokaryotic transcriptional regulators that controls the expression of genes involved in the anaerobic degradation of benzoate. We have shown that BzdR consists of an N-terminal DNA-binding domain connected through a linker to a C-terminal effector-binding domain that shows significant identity to the shikimate kinase (SK). The construction of active synthetic BzdR-like regulators by fusing the DNA-binding domain of BzdR to the Escherichia coli SKI protein strongly supports the notion that an ancestral SK domain could have been involved in the evolutionary origin of BzdR. The loss of the enzymatic activity of the ancestral SK domain was essential for it to evolve as a regulatory domain in the current BzdR protein. This work also supports the view that enzymes precede the emergence of the regulatory systems that may control their expression.