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.

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.

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.

Conformational studies of RGDechi peptide by natural-abundance NMR spectroscopy.

Integrins are heterodimeric cell-surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin-inhibitor drugs have been developed for integrin-based medical applications, the structural determinants modulating integrin-ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for αv ß3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of αv ß3 by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of αv ß3 integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance 15 N and 13 C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.

Binding mode of AIF(370-394) peptide to CypA: insights from NMR, label-free and molecular docking studies.

The complex formation between the proteins apoptosis-inducing factor (AIF) and cyclophilin A (CypA) following oxidative stress in neuronal cells has been suggested as a main target for reverting ischemia-stroke damage. Recently, a peptide encompassing AIF residues 370-394 has been developed to target the AIF-binding site on CypA, to prevent the association between the two proteins and suppress glutamate-induced cell death in neuronal cells. Using a combined approach based on NMR spectroscopy, synthesis and in vitro testing of all Ala-scan mutants of the peptide and molecular docking/molecular dynamics, we have generated a detailed model of the AIF (370-394)/CypA complex. The model suggests us that the central region of the peptide spanning residues V374-K384 mostly interacts with the protein and that for efficient complex inhibition and preservation of CypA activity, it is bent around amino acids F46-G75 of the protein. The model is consistent with experimental data also from previous works and supports the concept that the peptide does not interfere with other CypA activities unrelated to AIF activation; therefore, it may serve as an ideal template for generating future non-peptidic antagonists.

Insights into the anticancer properties of the first antimicrobial peptide from Archaea.

BACKGROUND: The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. METHODS: Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. RESULTS: It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. CONCLUSIONS: VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. GENERAL SIGNIFICANCE: VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

Structural and biochemical insights of CypA and AIF interaction.

The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370-394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases.

The interaction of heme with plakortin and a synthetic endoperoxide analogue: new insights into the heme-activated antimalarial mechanism.

In the present work we performed a combined experimental and computational study on the interaction of the natural antimalarial endoperoxide plakortin and its synthetic analogue 4a with heme. Obtained results indicate that the studied compounds produce reactive carbon radical species after being reductively activated by heme. In particular, similarly to artemisinin, the formation of radicals prone to inter-molecular reactions should represent the key event responsible for Plasmodium death. To our knowledge this is the first experimental investigation on the reductive activation of simple antimalarial endoperoxides (1,2-dioxanes) by heme and results were compared to the ones previously obtained from the reaction with FeCl2. The obtained experimental data and the calculated molecular interaction models represent crucial tools for the rational optimization of our promising class of low-cost synthetic antimalarial endoperoxides.

Synthetic waveforms of axial motion in a borehole with drill string.

This work studies the wave motion in a fluid-filled borehole in the presence of drill string and geological formation. The synthetic waveforms are obtained by a three-dimensional axis-symmetric full-wave numerical simulation in a two-dimensional multi-domain where the medium is uniform with respect to the azimuth. The discretization is performed in cylindrical coordinates. In order to simulate the waves at the origin (axis of the polar radius), a very small radius is used to avoid the singularity. The free-surface and rigid boundary conditions are tested and it is shown that the rigid one constitutes the best approximation. The simulations provide the amplitude distribution and motion diagrams in the borehole vertical cross-sections and at the outer boundary, away from the borehole. Propagation in the presence of hard and soft formations is analysed. The dispersion, the amplitude, and the orbital polarization of the modes excited by a point source acting in the fluid inside a drillstring are considered and examples of comparison with literature results obtained using multi-modal analysis are shown. The proposed approach is more general than the multi-modal analysis, since it allows for arbitrary variations of the properties in the plane of symmetry.

FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay: New Internally Quenched Fluorogenic Substrates for High-Throughput Screening.

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.

The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties.

A Combined NMR and Computational Approach to Determine the RGDechi-hCit-αv ß3 Integrin Recognition Mode in Isolated Cell Membranes.

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, αv ß3 and αv ß5 integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively αv ß3 integrin both in vitro and in vivo. High-resolution molecular details of the selective αv ß3 recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into αv ß3 molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for αv ß5 integrin.

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx: a novel peculiar member of the winged helix-turn-helix transcription factor family.

The hybrid plasmid-virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix-turn-helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog.

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain.

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the ~30 amino acid eukaryotic zinc finger motif in which a ß-sheet and an α-helix are clustered around a zinc ion by various combinations of four ligands. The prokaryotic zinc finger domain (found in the Ros protein from Agrobacterium tumefaciens) is different from the eukaryotic counterpart as it consists of 58 amino acids arranged in a ßßßαα topology stabilized by a 15-residue hydrophobic core. Also, this domain tetrahedrally coordinates zinc and unfolds in the absence of the metal ion. The characterization of proteins belonging to the Ros homologs family has however shown that the prokaryotic zinc finger domain can overcome the metal requirement to achieve the same fold and DNA-binding activity. In the present work, two zinc-lacking Ros homologs (Ml4 and Ml5 proteins) have been thoroughly characterized using bioinformatics, biochemical and NMR techniques. We show how in these proteins a network of hydrogen bonds and hydrophobic interactions surrogate the zinc coordination role in the achievement of the same functional fold.

Deciphering the zinc coordination properties of the prokaryotic zinc finger domain: The solution structure characterization of Ros87 H42A functional mutant.

The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys2His2 zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first α-helix. At the same time, the second helix appears more mobile in the µs-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type.

Molecular basis of the PED/PEA15 interaction with the C-terminal fragment of phospholipase D1 revealed by NMR spectroscopy.

PED/PEA15 is a small protein involved in many protein-protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion. The C-terminal portion (residues 712-1074) of PLD1, named D4, is still able to interact with PED/PEA15. In this study we characterized, by means of NMR spectroscopy, the molecular interaction of PED/PEA15 with D4α, a smaller region of D4, encompassing residues 712-818, shown to have the same affinity for PED/PEA15 and to induce the same effects as D4 in PED/PEA15-overexpressing cells. Chemical shift perturbation (CSP) studies allowed to define D4α binding site of PED/PEA15 and to identify a smaller region likely affected by an allosteric effect. Moreover, ELISA-like experiments showed that three 20-mer overlapping synthetic peptides, covering the 762-801 region of D4α, strongly inhibit PED/PEA15-D4α interaction through their binding to PED/PEA15 with KDs in low micromolar range. Finally, molecular details of the interaction of PED/PEA15 with one of the three peptides have been revealed by CSP and saturation transfer difference (STD) analyses.

Targeting zinc finger domains with small molecules: solution structure and binding studies of the RanBP2-type zinc finger of RBM5.

The RNA binding motif protein 5 (RBM5), also known as Luca15 or H37, is a component of prespliceosomal complexes that regulates the alternative splicing of several mRNAs, such as Fas and caspase-2. The RBM5 gene is located at the 2p21.3 chromosomal region, which is strongly associated with lung cancer and many other cancers. Both increased and decreased levels of RBM5 can play a role in tumor progression. In particular, downregulation of rbm5 is involved in lung cancer and other cancers upon Ras activation, and, also, represents a molecular signature associated with metastasis in various solid tumors. On the other hand, upregulation of RBM5 occurs in breast and ovarian cancer. Moreover, RBM5 was also found to be involved in the early stage of the HIV-1 viral cycle, representing a potential target for the treatment of the HIV-1 infection. While the molecular basis for RNA recognition and ubiquitin interaction has been structurally characterized, small molecules binding this zinc finger (ZF) domain that might contribute to characterizing their activity and to the development of potential therapeutic agents have not yet been reported. Using an NMR screening of a fragment library we identified several binders and the complex of the most promising one, compound 1, with the RBM5 ZF1 was structurally characterized in solution. Interestingly, the binding mechanism reveals that 1 occupies the RNA binding pocket and is therefore able to compete with the RNA to bind RBM5 RanBP2-type ZF domain, as indicated by NMR studies.

Targeting metalloproteins by fragment-based lead discovery.

It has been estimated that nearly one-third of functional proteins contain a metal ion. These constitute a wide variety of possible drug targets including metalloproteinases, dehydrogenases, oxidoreductases, hydrolases, deacetylases, or many others in which the metal ion is either of catalytic or of structural nature. Despite the predominant role of a metal ion in so many classes of drug targets, current high-throughput screening techniques do not usually produce viable hits against these proteins, likely due to the lack of proper metal-binding pharmacophores in the current screening libraries. Herein, we describe a novel fragment-based drug discovery approach using a metal-targeting fragment library that is based on a variety of distinct classes of metal-binding groups designed to reliably anchor the fragments at the target's metal ions. We show that the approach can effectively identify novel, potent and selective agents that can be readily developed into metalloprotein-targeted therapeutics.

NMR backbone dynamics studies of human PED/PEA-15 outline protein functional sites.

PED/PEA-15 (phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes) is a ubiquitously expressed protein and a key regulator of cell growth and glucose metabolism. PED/PEA-15 mediates both homotypic and heterotypic interactions and is constituted by an N-terminal canonical death effector domain and a C-terminal tail. In the present study, the backbone dynamics of PED/PEA-15 via (15)N R(1) and R(2) and steady-state [(1)H]-(15)N NOE measurements is reported. The dynamic parameters were analyzed using both Lipari-Szabo model-free formalism and a reduced spectral density mapping approach. The results obtained define a polar and charged surface of the death effector domain characterized by internal motions in the micro- to millisecond timescale, which is crucial for the multiple heterotypic functional protein-protein interactions in which PED/PEA-15 is involved. The present study contributes to a better understanding of the molecular basis of the PED/PEA-15 functional interactions and provides a more detailed surface for the design and development of PED/PEA-15 binders.