Francesco Oteri

Structure of nanoscale truncated octahedral DNA cages: variation of single-stranded linker regions and influence on assembly yields.

The assembly, structure, and stability of DNA nanocages with the shape of truncated octahedra have been studied. The cages are composed of 12 double-stranded B-DNA helices interrupted by single-stranded linkers of thymidines of varying length that constitute the truncated corners of the structure. The structures assemble with a high efficiency in a one-step procedure, compared to previously published structures of similar complexity. The structures of the cages were determined by small-angle X-ray scattering. With increasing linker length, there is a systematic increase of the cage size and decrease of the twist angle of the double helices with respect to the symmetry planes of the cage structure. In the present study, we demonstrate the length of the single-stranded linker regions, which impose a certain degree of flexibility to the structure, to be the important determinant for efficient assembly. The linker length can be decreased to three thymidines without affecting assembly yield or the overall structural characteristics of the DNA cages. A linker length of two thymidines represents a sharp cutoff abolishing cage assembly. This is supported by energy minimization calculations suggesting substantial hydrogen bond deformation in a cage with linkers of two thymidines.

Conjugated eicosapentaenoic acid inhibits human topoisomerase IB with a mechanism different from camptothecin

Conjugated eicosapentaenoic acid (cEPA) has been found to have antitumor effects which has been ascribed to their ability to inhibit DNA topoisomerases and DNA polymerases. We here show that cEPA inhibits the catalytic activity of human topoisomerase I, but unlike camptothecin it does not stabilize the cleavable complex, indicating a different mechanism of action. cEPA inhibits topoisomerase by impeding the catalytic cleavage of the DNA substrate as demonstrated using specific oligonucleotide substrates, and prevents the stabilization of the cleavable complex by camptothecin. Preincubation of the inhibitor with the enzyme is required to obtain complete inhibition. Molecular docking simulations indicate that the preferred cEPA binding site is proximal to the active site with the carboxylic group strongly interacting with the positively charged K443 and K587. Taken together the results indicate that cEPA inhibitor does not prevent DNA binding but inhibits DNA cleavage, binding in a region close to the topoisomerase active site.

Deciphering the structural properties that confer stability to a DNA nanocage.

A DNA nanocage has been recently characterized by small-angle X-ray scattering (SAXS) and cryo-transmission electron microscopy as a DNA octahedron having a central cavity larger than the apertures in the surrounding DNA lattice. Starting from the SAXS data, a DNA nanocage has been modeled and simulated by classical molecular dynamics to evaluate in silico its structural properties and stability. Global properties, principal component analysis, and DNA geometrical parameters, calculated along the entire trajectory, indicate that the cage is stable and that the B-DNA conformation, also if slightly distorted, is maintained for all the simulation time. Starting from the initial model, the nanocage scaffold undergoes a contraction of the thymidine strands, connecting the DNA double helices, suggesting that the length of the thymidine strands is a crucial aspect in the modulation of the nanocage stability. A comparison of the average structure as obtained from the simulation shows good agreement with the SAXS experimental data.

Characterization of a novel CYP2C9 gene mutation and structural bioinformatic protein analysis in a warfarin hypersensitive patient

Warfarin (coumadin) is a worldwide-prescribed anticoagulant for the long-term treatment and prevention of thromboembolic events, presenting a great interindividual variability in the required dose. It is known that both environmental and genetic factors influence the dose necessary for the therapeutic effect. Herein we describe a pharmacogenetic study conducted on an Italian patient with warfarin hypersensitivity, who required a very low dosage to achieve therapeutic anticoagulation effect. We genotyped common polymorphisms in VKORC1, CYP2C9, and CYP4F2 genes, known to be involved in warfarin dosing. As the patient resulted in a mixture of low-dosing and high-dosing polymorphic variants, we searched for rare mutations by direct sequencing of the same genes. We identified in the CYP2C9 gene, a novel mutation in heterozygote status, c.374G>T, which produces the Arg125Leu substitution. We have observed, through an electrostatic analysis, that the new mutation produces an electrostatic alteration on the cytochrome surface.

High warfarin sensitivity in carriers of CYP2C9*35 is determined by the impaired interaction with P450 oxidoreductase

Cytochrome P450 2C9 (CYP2C9) metabolizes many clinically important drugs including warfarin and diclofenac. We have recently reported a new allelic variant, CYP2C9*35, found in a warfarin hypersensitive patient with Arg125Leu and Arg144Cys mutations. Here, we have investigated the molecular basis for the functional consequences of these polymorphic changes. CYP2C9.1 and CYP2C9-Arg144Cys expressed in human embryonic kidney 293 cells effectively metabolized both S-warfarin and diclofenac in NADPH-dependent reactions, whereas CYP2C9-Arg125Leu or CYP2C9.35 were catalytically silent. However, when NADPH was replaced by a direct electron donor to CYPs, cumene hydroperoxide, hereby bypassing the CYP oxidoreductase (POR), all variant enzymes were active, indicating unproductive interactions between CYP2C9.35 and POR. In silico analysis revealed a decrease of the electrostatic potential of CYP2C9-Arg125Leu-POR interacting surface and the loss of stabilizing salt bridges between these proteins. In conclusion, our data strongly suggest that the Arg125Leu mutation in CYP2C9.35 prevents CYP2C9-POR interactions resulting in the absence of NADPH-dependent CYP2C9-catalyzed activity in vivo, thus influencing the warfarin sensitivity in the carriers of this allele.

Structural-dynamical investigation of the ZnuA histidine-rich loop: involvement in zinc management and transport.

Comparative homology modelling techniques have been used to model the protein ZnuA from Salmonella enterica serovar Typhimurium using the 3D structure of the homologous protein from Escherichia coli. These two-domain proteins bind one Zn2+ atom, with high affinity, in the inter-domain cleft and possess a histidine-rich loop in the N-terminal domain. Alternative structures of the ZnuA histidine-rich loop, never resolved by the X-ray diffraction method, have been modelled. A model of the apo form, one with the histidine-rich loop deleted and two alternative structures with a second zinc ion bound to the histidine-rich loop, have been generated. In all the modelled proteins, investigated through molecular dynamics simulation, the histidine-rich loop is highly mobile and its fluctuations are correlated to the ligand stability observed in the zinc sites. Based on the plasticity of the histidine-rich loop and its significant effects on protein mobility a possible role in the capture and/or transfer of the zinc ions has been suggested.

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.

MD Simulations of Papillomavirus DNA-E2 Protein Complexes Hints at a Protein Structural Code for DNA Deformation

The structural dynamics of the DNA binding domains of the human papillomavirus strain 16 and the bovine papillomavirus strain 1, complexed with their DNA targets, has been investigated by modeling, molecular dynamics simulations, and nuclear magnetic resonance analysis. The simulations underline different dynamical features of the protein scaffolds and a different mechanical interaction of the two proteins with DNA. The two protein structures, although very similar, show differences in the relative mobility of secondary structure elements. Protein structural analyses, principal component analysis, and geometrical and energetic DNA analyses indicate that the two transcription factors utilize a different strategy in DNA recognition and deformation. Results show that the protein indirect DNA readout is not only addressable to the DNA molecule flexibility but it is finely tuned by the mechanical and dynamical properties of the protein scaffold involved in the interaction.

Large-scale identification of coevolution signals across homo-oligomeric protein interfaces by direct coupling analysis

Significance Protein–protein interactions are important to all facets of life, but their experimental and computational characterization is arduous and frequently of uncertain outcome. The current study demonstrates both the power and limitation to study protein interactions by utilizing sophisticated statistical inference technology to derive protein contacts from available sequence databases, more precisely from the coevolution between residues, that are in contact across the interaction interface of two proteins. By studying homo-oligomeric protein interactions, the current study expands from anecdotal evidence of the performance of this technology to systematic evidence of its value across close to 2,000 interacting protein families. Proteins have evolved to perform diverse cellular functions, from serving as reaction catalysts to coordinating cellular propagation and development. Frequently, proteins do not exert their full potential as monomers but rather undergo concerted interactions as either homo-oligomers or with other proteins as hetero-oligomers. The experimental study of such protein complexes and interactions has been arduous. Theoretical structure prediction methods are an attractive alternative. Here, we investigate homo-oligomeric interfaces by tracing residue coevolution via the global statistical direct coupling analysis (DCA). DCA can accurately infer spatial adjacencies between residues. These adjacencies can be included as constraints in structure prediction techniques to predict high-resolution models. By taking advantage of the ongoing exponential growth of sequence databases, we go significantly beyond anecdotal cases of a few protein families and apply DCA to a systematic large-scale study of nearly 2,000 Pfam protein families with sufficient sequence information and structurally resolved homo-oligomeric interfaces. We find that large interfaces are commonly identified by DCA. We further demonstrate that DCA can differentiate between subfamilies with different binding modes within one large Pfam family. Sequence-derived contact information for the subfamilies proves sufficient to assemble accurate structural models of the diverse protein-oligomers. Thus, we provide an approach to investigate oligomerization for arbitrary protein families leading to structural models complementary to often-difficult experimental methods. Combined with ever more abundant sequential data, we anticipate that this study will be instrumental to allow the structural description of many heteroprotein complexes in the future.

Molecular mechanism of statin-mediated LOX-1 inhibition

Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins.