Descripción: The phytochemical study of two species of Jaborosa caulescens (var. caulescens and var. bipinnatifida) yielded the four new withanolides 1-4. The structures of the new compounds were determined using a combination of spectroscopic techniques (including 1D and 2D NMR) and Molecular Modeling.

Descripción: Frataxin (FXN) is an α/β protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90-195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90-195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90-195 and hFXN90-210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function. © 2012 Roman et al.

Descripción: We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mM concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.

Descripción: 2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg2+ (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5mMATP. Remarkably, the withdrawal of ATP or Mg2+ brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter ∼30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of β-sheets with the concurrent decrease in the content of α-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce α-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg129 and Arg152, are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDLcontaining proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and thatCHDLdomains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: A highly sulfated 3-linked β-arabinan (Ab1) with arabinose in the pyranose form was obtained from green seaweed Codium vermilara (Bryopsidales). It comprised major amounts of units sulfated on C-2 and C-4 and constitutes the first polysaccharide of this type isolated in the pure form and fully characterized. Ab1 showed anticoagulant activity by global coagulation tests. Less sulfated arabinans obtained from the same seaweed have less or no activity. Ab1 exerts its activity through direct and indirect (antithrombin- and heparin cofactor II-mediated) inhibition of thrombin. Direct thrombin inhibition was studied in detail. By native PAGE, it was possible to detect formation of a complex between Ab1 and human thrombin (HT). Ab1 binding to HT was measured by fluorescence spectroscopy. CD spectra of the Ab1 complex suggested that ligand binding induced a small conformational change on HT. Ab1-thrombin interactions were studied by molecular dynamic simulations using the persulfated octasaccharide as model compound. Most carbohydrate-protein contacts would occur by interaction of sulfate groups with basic amino acid residues on the surface of the enzyme, more than 60% of them being performed by the exosite 2-composing residues. In these interactions, the sulfate groups on C-2 were shown to interact more intensely with the thrombin structure. In contrast, the disulfated oligosaccharide does not promote major conformational modifications at the catalytic site when complexed to exosite 1. These results show that this novel pyranosic sulfated arabinan Ab1 exerts its anticoagulant activity by a mechanism different from those found previously for other sulfated polysaccharides and glycosaminoglycans. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol-1), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol-1 from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: Background: Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism. Methodology/Principal Findings: Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric "Z-nucleus" after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 μM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a "conformation editing" mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route. Conclusion: We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions. © 2012 Smal et al.

Descripción: Background: Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts as a transcriptional modulator and exerts a key role in viral DNA replication. Thus, E2 constitutes an attractive target for developing antiviral agents. E2 is a homodimeric protein that interacts with the DNA target through an α-helix of each monomer. However, a peptide corresponding to the DNA recognition helix of HPV-16 E2 binds DNA with lower affinity than its full-length DNA binding domain. Therefore, in an attempt to promote the DNA binding of the isolated peptide, we have designed a conjugate compound of the E2 α-helix peptide and a derivative of the antibiotic distamycin, which involves simultaneous minor- and major-groove interactions. Methodology/Principal Findings: An E2 α-helix peptide-distamycin conjugate was designed and synthesized. It was characterized by NMR and CD spectroscopy, and its DNA binding properties were investigated by CD, DNA melting and gel shift experiments. The coupling of E2 peptide with distamycin does not affect its structural properties. The conjugate improves significantly the affinity of the peptide for specific DNA. In addition, stoichiometric amounts of specific DNA increase meaningfully the helical population of the peptide. The conjugate enhances the DNA binding constant 50-fold, maintaining its specificity. Conclusions/Significance: These results demonstrate that peptide-distamycin conjugates are a promising tool to obtain compounds that bind the E2 target DNA-sequences with remarkable affinity and suggest that a bipartite major/minor groove binding scaffold can be a useful approach for therapeutic treatment of HPV infection. © 2011 Wetzler et al.