Structural characterization of PHOX2B and its DNA interaction shed light on the molecular basis of the +7Ala variant pathogenicity in CCHS.

An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome (CCHS). Recent studies demonstrated that the alanine tract expansion influences PHOX2B folding and activity. Therefore, structural information on PHOX2B is an important target for obtaining clues to elucidate the insurgence of the alanine expansion-related syndrome and also for defining a viable therapy. Here we report by NMR spectroscopy the structural characterization of the homeodomain (HD) of PHOX2B and HD + C-terminus PHOX2B protein, free and in the presence of the target DNA. The obtained structural data are then exploited to obtain a structural model of the PHOX2B-DNA interaction. In addition, the variant +7Ala, responsible for one of the most frequent forms of the syndrome, was analysed, showing different conformational proprieties in solution and a strong propensity to aggregation. Our data suggest that the elongated poly-alanine tract would be related to disease onset through a loss-of-function mechanism. Overall, this study paves the way for the future rational design of therapeutic drugs, suggesting as a possible therapeutic route the use of specific anti-aggregating molecules capable of preventing variant aggregation and possibly restoring the DNA-binding activity of PHOX2B.

Molecular interactions between a diphenyl scaffold and PED/PEA15: Implications for type II diabetes therapeutics targeting PED/PEA15 - Phospholipase D1 interaction.

In a recent study, we have identified BPH03 as a promising scaffold for the development of compounds aimed at modulating the interaction between PED/PEA15 (Phosphoprotein Enriched in Diabetes/Phosphoprotein Enriched in Astrocytes 15) and PLD1 (phospholipase D1), with potential applications in type II diabetes therapy. PED/PEA15 is known to be overexpressed in certain forms of diabetes, where it binds to PLD1, thereby reducing insulin-stimulated glucose transport. The inhibition of this interaction reestablishes basal glucose transport, indicating PED as a potential target of ligands capable to recover glucose tolerance and insulin sensitivity. In this study, we employ computational methods to provide a detailed description of BPH03 interaction with PED, evidencing the presence of a hidden druggable pocket within its PLD1 binding surface. We also elucidate the conformational changes that occur during PED interaction with BPH03. Moreover, we report new NMR data supporting the in-silico findings and indicating that BPH03 disrupts the PED/PLD1 interface displacing PLD1 from its interaction with PED. Our study represents a significant advancement toward the development of potential therapeutics for the treatment of type II diabetes.

Inclusions of Pesticides by ß-Cyclodextrin in Solution and Solid State: Chlorpropham, Monuron, and Propanil.

Persistence and degradation are important factors in determining the safe use of such synthetic products, and numerous studies have been addressed to develop pesticide remediation methods aimed at ameliorating these features. In this frame, the use of different cyclodextrins (CDs) molecules has attracted considerable attention due to their well-known non-toxic nature, limited environmental impact, and capability to reduce the environmental and health risks of pesticides. CDs appear to be a valuable tool for the elimination of pesticides from polluted areas as well as for better pesticide formulations that positively influence their hydrolysis or degradation. The present work investigates the interaction between ß-cyclodextrins and three commonly used pesticides (i.e., chlorpropham, monuron, and propanil) both in solution and in the solid state by means of UV-Vis, FT-IR, and X-ray powder diffractometry. We show that such interactions result in all three cases in the formation of inclusion complexes with a 1:1 stoichiometry and binding constants (Kb) of 369.9 M-1 for chlorpropham, 292.3 M-1 for monuron, and 298.3 M-1 for propanil. We also report the energy-minimized structures in silico for each complex. Our data expand and complement the available literature data in indicating CDs as a low-cost and very effective tool capable of modulating the properties that determine the environmental fate of pesticides.

Copper (I) or (II) Replacement of the Structural Zinc Ion in the Prokaryotic Zinc Finger Ros Does Not Result in a Functional Domain.

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper's interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.

High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations.

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvß3 without cross reacting with αvß5 and αIIbß3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvß3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions.

Structural and dynamical determinants of a ß-sheet-enriched intermediate involved in amyloid fibrillar assembly of human prion protein.

The conformational conversion of the cellular prion protein (PrPC) into a misfolded, aggregated and infectious scrapie isoform is associated with prion disease pathology and neurodegeneration. Despite the significant number of experimental and theoretical studies the molecular mechanism regulating this structural transition is still poorly understood. Here, via Nuclear Magnetic Resonance (NMR) methodologies we investigate at the atomic level the mechanism of the human HuPrP(90-231) thermal unfolding and characterize the conformational equilibrium between its native structure and a ß-enriched intermediate state, named ß-PrPI. By comparing the folding mechanisms of metal-free and Cu2+-bound HuPrP(23-231) and HuPrP(90-231) we show that the coupling between the N- and C-terminal domains, through transient electrostatic interactions, is the key molecular process in tuning long-range correlated µs-ms dynamics that in turn modulate the folding process. Moreover, via thioflavin T (ThT)-fluorescence fibrillization assays we show that ß-PrPI is involved in the initial stages of PrP fibrillation, overall providing a clear molecular description of the initial phases of prion misfolding. Finally, we show by using Real-Time Quaking-Induced Conversion (RT-QuIC) that the ß-PrPI acts as a seed for the formation of amyloid aggregates with a seeding activity comparable to that of human infectious prions.

Switching the N-Capping Region from all-L to all-D Amino Acids in a VEGF Mimetic Helical Peptide.

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated.

Formulation of Solid Lipid Nanoparticles Loaded with Nociceptin/Orphanin FQ (N/OFQ) and Characterization in a Murine Model of Airway Hyperresponsiveness.

Asthma is characterized by chronic inflammation and a variable degree of airway hyperresponsiveness (AHR). Our previous papers documented a role for Nociceptin/Orphanin FQ (N/OFQ) and its receptor N/OFQ peptide (NOP) in AHR. Therefore, the aim of this study was to improve the bioavailability of N/OFQ by developing solid lipid nanoparticles (SLNs). N/OFQ-loaded SLNs were prepared by the Quasi Emulsion Solvent Diffusion (QESD) technique and then characterized. Brown Norway rats were sensitized to ovalbumin (OVA) and treated with an intratracheal administration of saline solution or N/OFQ-SLN. Then, 24 h after the last challenge, functional histological and molecular evaluations were performed. SLNs showed a mean diameter of 233 ± 0.03 nm, a polydispersity index (PDI) value around 0.28 ± 0.02 and a drug release percentage of 84.3. The in vitro release of N/OFQ from SLNs showed that the release of the peptide starts already after two hours of incubation. Animals receiving N/OFQ-SLN showed a significative decrease in allergen-induced AHR compared to the control group. These results showed the positive effects of N/OFQ-SLNs on the inflammatory process and on the mechanical properties of the airways, suggesting that the innovative nanotechnological approach may be therapeutically beneficial for asthmatic patients.

Modulation of the 20S Proteasome Activity by Porphyrin Derivatives Is Steered through Their Charge Distribution.

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure-activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg-Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.

Structural characterization of the thermal unfolding pathway of human VEGFR1 D2 domain.

Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high-resolution thermodynamic approach that combines differential scanning calorimetry with atomic-level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a 'folding nucleus' induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.

Screening a Molecular Fragment Library to Modulate the PED/PEA15-Phospholipase D1 Interaction in Cellular Lysate Environments.

The overexpression of PED/PEA15, the phosphoprotein enriched in diabetes/phosphoprotein enriched in the astrocytes 15 protein (here referred simply to as PED), observed in some forms of type II diabetes, reduces the transport of insulin-stimulated glucose by binding to the phospholipase D1 (PLD1). The inhibition of the PED/PLD1 interaction was shown to restore basal glucose transport, indicating PED as a pharmacological target for the development of drugs capable of improving insulin sensitivity and glucose tolerance. We here report the identification and selection of PED ligands by means of NMR screening of a library of small organic molecules, NMR characterization of the PED/PLD1 interaction in lysates of cells expressing PLD1, and modulation of such interactions using BPH03, the best selected ligand. Overall, we complement the available literature data by providing detailed information on the structural determinants of the PED/PLD1 interaction in a cellular lysate environment and indicate BPH03 as a precious scaffold for the development of novel compounds that are able to modulate such interactions with possible therapeutic applications in type II diabetes.

Probing the helical stability in a VEGF-mimetic peptide.

The analysis of the forces governing helix formation and stability in peptides and proteins has attracted considerable interest in order to shed light on folding mechanism. We analyzed the role of hydrophobic interaction, steric hindrance and chain length on i, i + 3 position in QK peptide, a VEGF mimetic helical peptide. We focused on position 10 of QK, occupied by a leucine, as previous studies highlighted the key role of the Leu7-Leu10 interaction in modulating the helix formation and inducing an unusual thermodynamic stability. Leu10 has been replaced by hydrophobic amino acids with different side-chain length, hydrophobicity and steric hindrance. Ten peptides were, hence, synthesized and analyzed combining circular dichroism, calorimetry and NMR spectroscopy. We found that helical content and thermal stability of peptide QK changed when Leu10 was replaced. Interestingly, we observed that the changes in the helical content and thermal stability were not always correlated and they depend on the type of interaction (strength and geometry) that could be established between Leu7 and the residue in position 10.

Design, Optimization, and Structural Characterization of an Apoptosis-Inducing Factor Peptide Targeting Human Cyclophilin A to Inhibit Apoptosis Inducing Factor-Mediated Cell Death.

Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a ß-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features.

A novel approach for studying receptor-ligand interactions on living cells surface by using NUS/T1ρ-NMR methodologies combined with computational techniques: The RGDechi15D-αvß5 integrin complex.

Structural investigations of receptor-ligand interactions on living cells surface by high-resolution Nuclear Magnetic Resonance (NMR) are problematic due to their short lifetime, which often prevents the acquisition of experiments longer than few hours. To overcome these limitations, we developed an on-cell NMR-based approach for exploring the molecular determinants driving the receptor-ligand recognition mechanism under native conditions. Our method relies on the combination of high-resolution structural and dynamics NMR data with Molecular Dynamics simulations and Molecular Docking studies. The key point of our strategy is the use of Non Uniform Sampling (NUS) and T1ρ-NMR techniques to collect atomic-resolution structural and dynamics information on the receptor-ligand interactions with living cells, that can be used as conformational constraints in computational studies. In fact, the application of these two NMR methodologies allows to record spectra with high S/N ratio and resolution within the lifetime of cells. In particular, 2D NUS [1H-1H] trNOESY spectra are used to explore the ligand conformational changes induced by receptor binding; whereas T1ρ-based experiments are applied to characterize the ligand binding epitope by defining two parameters: T1ρ Attenuation factor and T1ρ Binding Effect. This approach has been tested to characterize the molecular determinants regulating the recognition mechanism of αvß5-integrin by a selective cyclic binder peptide named RGDechi15D. Our data demonstrate that the developed strategy represents an alternative in-cell NMR tool for studying, at atomic resolution, receptor-ligand recognition mechanism on living cells surface. Additionally, our application may be extremely useful for screening of the interaction profiling of drugs with their therapeutic targets in their native cellular environment.

Metabolic and conformational stabilization of a VEGF-mimetic beta-hairpin peptide by click-chemistry.

HPLW is a Vascular Endothelial Growth Factor (VEGF)-mimicking beta-hairpin peptide endowed of proangiogenic effect and showing valuable biomedical application in the proangiogenic therapy. However, the translational potential of HPLW is limited by its low metabolic stability, which would shorten the in vivo efficacy of the molecule. Here, we developed a peptide analog of HPLW, named HPLW2, that retains the structural and biological properties of the original peptide but features an impressive resistance to degradation by human serum proteases. HPLW2 was obtained by covalently modifying the chemical structure of the peptide with molecular tools known to impart protease resistance. Notably, the peptide was cyclized by installing an interstrand triazole bridge through Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction. HPLW2 appears as a novel and promising drug candidate with potential biomedical application in the proangiogenic therapy as a low molecular weight drug, alternative to the use of VEGF. Our work points out the utility of the interstrand triazole bridge as effective chemical platform for the conformational and metabolic stabilization of beta-hairpin bioactive peptides.

The change of conditions does not affect Ros87 downhill folding mechanism.

Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. Downhill folders are estimated to be rather rare in nature as they miss an energetically substantial folding barrier that can protect against aggregation and proteolysis. We have previously demonstrated that the prokaryotic zinc finger protein Ros87 shows a bipartite folding/unfolding process in which a metal binding intermediate converts to the native structure through a delicate barrier-less downhill transition. Significant variation in folding scenarios can be detected within protein families with high sequence identity and very similar folds and for the same sequence by varying conditions. For this reason, we here show, by means of DSC, CD and NMR, that also in different pH and ionic strength conditions Ros87 retains its partly downhill folding scenario demonstrating that, at least in metallo-proteins, the downhill mechanism can be found under a much wider range of conditions and coupled to other different transitions. We also show that mutations of Ros87 zinc coordination sphere produces a different folding scenario demonstrating that the organization of the metal ion core is determinant in the folding process of this family of proteins.

Substitution of the Native Zn(II) with Cd(II), Co(II) and Ni(II) Changes the Downhill Unfolding Mechanism of Ros87 to a Completely Different Scenario.

The structural effects of zinc replacement by xenobiotic metal ions have been widely studied in several eukaryotic and prokaryotic zinc-finger-containing proteins. The prokaryotic zinc finger, that presents a bigger ßßßαα domain with a larger hydrophobic core with respect to its eukaryotic counterpart, represents a valuable model protein to study metal ion interaction with metallo-proteins. Several studies have been conducted on Ros87, the DNA binding domain of the prokaryotic zinc finger Ros, and have demonstrated that the domain appears to structurally tolerate Ni(II), albeit with important structural perturbations, but not Pb(II) and Hg(II), and it is in vitro functional when the zinc ion is replaced by Cd(II). We have previously shown that Ros87 unfolding is a two-step process in which a zinc binding intermediate converts to the native structure thorough a delicate downhill folding transition. Here, we explore the folding/unfolding behaviour of Ros87 coordinated to Co(II), Ni(II) or Cd(II), by UV-Vis, CD, DSC and NMR techniques. Interestingly, we show how the substitution of the native metal ion results in complete different folding scenarios. We found a two-state unfolding mechanism for Cd-Ros87 whose metal affinity Kd is comparable to the one obtained for the native Zn-Ros87, and a more complex mechanism for Co-Ros87 and Ni-Ros87, that show higher Kd values. Our data outline the complex cross-correlation between the protein-metal ion equilibrium and the folding mechanism proposing such an interplay as a key factor in the proper metal ion selection by a specific metallo-protein.

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States.

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.

Selective Targeting of αvß5 Integrin in HepG2 Cell Line by RGDechi15D Peptide.

Recently, the research community has become increasingly concerned with the receptor αvß5, a member of the well-known integrin family. Different ongoing studies have evidenced that αvß5 integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. Remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. In this scenario, the availability of a selective αvß5 antagonist without 'off-target' protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. We recently reported a cyclic peptide, RGDechi15D, obtained by structure-activity studies. To our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvß5 integrin and not cross react with αvß3. Here we demonstrated the ability of the peptide to diminish both adhesion and invasion of HepG2 cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the PI3K pathway. The peptide, also decreases the formation of new vessels in endothelial cells. Taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma.

Zinc Fingers.

Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. While their amino acid composition varies from one domain to the other, a shared feature is the coordination of a zinc ion, with a structural role, by a different combination of cysteines and histidines. The classical zinc finger domain (also called Cys2His2) that represents the most common class, uses two cysteines and two histidines to coordinate the metal ion, and forms a compact ßßα architecture consisting in a ß-sheet and an α-helix. GAG-knuckle resembles the classical ZF, treble clef and zinc ribbon are also well represented in the human genome. Zinc fingers are also present in prokaryotes. The first prokaryotic ZF domain found in the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. It shows a Cys2His2 metal ion coordination sphere and folds in a domain significantly larger than its eukaryotic counterpart arranged in a ßßßαα topology. Interestingly, this domain does not strictly require the metal ion coordination to achieve the functional fold. Here, we report what is known on the main classes of eukaryotic and prokarotic ZFs, focusing our attention to the role of the metal ion, the folding mechanism, and the DNA binding. The hypothesis of a horizontal gene transfer from prokaryotes to eukaryotes is also discussed.