Steric hindrance and structural flexibility shape the functional properties of a guanine-rich oligonucleotide.

Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers.

Aptamers: Functional-Structural Studies and Biomedical Applications 2.0.

As a follow-up to the previous Special Issue "Aptamers: Functional-Structural Studies and Biomedical Applications" [...].

Cisplatin Binding to Human Serum Transferrin: A Crystallographic Study.

The molecular mechanism of how human serum transferrin (hTF) recognizes cisplatin at the atomic level is still unclear. Here, we report the molecular structure of the adduct formed upon the reaction of hTF with cisplatin. Pt binds the side chain of Met256 (at the N-lobe), without altering the protein overall conformation.

Structural Insights into Protein-Aptamer Recognitions Emerged from Experimental and Computational Studies.

Aptamers are synthetic nucleic acids that are developed to target with high affinity and specificity chemical entities ranging from single ions to macromolecules and present a wide range of chemical and physical properties. Their ability to selectively bind proteins has made these compounds very attractive and versatile tools, in both basic and applied sciences, to such an extent that they are considered an appealing alternative to antibodies. Here, by exhaustively surveying the content of the Protein Data Bank (PDB), we review the structural aspects of the protein-aptamer recognition process. As a result of three decades of structural studies, we identified 144 PDB entries containing atomic-level information on protein-aptamer complexes. Interestingly, we found a remarkable increase in the number of determined structures in the last two years as a consequence of the effective application of the cryo-electron microscopy technique to these systems. In the present paper, particular attention is devoted to the articulated architectures that protein-aptamer complexes may exhibit. Moreover, the molecular mechanism of the binding process was analyzed by collecting all available information on the structural transitions that aptamers undergo, from their protein-unbound to the protein-bound state. The contribution of computational approaches in this area is also highlighted.

Aptamers: Functional-Structural Studies and Biomedical Applications.

Aptamers are synthetic molecules of different natures (mostly, DNA or RNA) that recognize a target molecule with high affinity and specificity [...].

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands.

Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.

Structure-Activity Relationship Study of a Potent α-Thrombin Binding Aptamer Incorporating Hexitol Nucleotides.

The replacement of one or more nucleotide residues in the potent α-thrombin-binding aptamer NU172 with hexitol-based nucleotides has been devised to study the effect of these substitutions on the physicochemical and functional properties of the anticoagulant agent. The incorporation of single hexitol nucleotides at the T9 and G18 positions of NU172 substantially retained the physicochemical features of the parent oligonucleotide, as a result of the biomimetic properties of the hexitol backbone. Importantly, the NU172-TH 9 mutant exhibited a higher binding affinity toward human α-thrombin than the native aptamer and an improved stability even after 24 h in 90 % human serum, with a significant increase in the estimated half-life. The anticoagulant activity of the modified oligonucleotide was also found to be slightly preferable to NU172. Overall, these results confirm the potential of hexitol nucleotides as biomimetic agents, while laying the foundations for the development of NU172-inspired α-thrombin-binding aptamers.

Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer.

Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer. The crystal structure of thrombin-NU172 complex reveals a bimodular duplex/quadruplex architecture for the aptamer, which binds thrombin exosite I through a highly complementary surface involving all three loops of the G-quadruplex module. Although the duplex domain does not interact directly with thrombin, the features of the duplex/quadruplex junction and the solution data on two newly designed NU172 mutants indicate that the duplex moiety is important for the optimization of the protein-ligand interaction and for the inhibition of the enzyme activity. Our work discloses the structural features determining the inhibition of thrombin by NU172 and put the basis for the design of mutants with improved properties.

Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers.

Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability.

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.

Probing the activity of diguanylate cyclases and c-di-GMP phosphodiesterases in real-time by CD spectroscopy.

Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3'-5' cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.

Effect of NaCl on the conformational stability of the thermophilic γ-glutamyltranspeptidase from Geobacillus thermodenitrificans: Implication for globular protein halotolerance.

The transpeptidation activity of γ-glutamyltranspeptidase from Geobacillus thermodenitrificans (GthGT) is negligible and the enzyme is highly thermostable. Here we have examined the effect of concentrated NaCl solutions on structure, stability, dynamics and enzymatic activity of GthGT. The protein exhibited hydrolytic activity over a broad range of NaCl concentrations. Even at 4.0M NaCl, GthGT retained more than 90% of the initial activity and showed unaltered fluorescence emission, secondary structure and acrylamide quenching on tryptophan fluorescence. Furthermore, at 2.8M and 4.0M NaCl the temperature-induced unfolding profiles are dramatically changed with large (>20°C) positive shifts in the denaturation temperature. These features make GthGT an ideal system to be used in industrial processes that require high temperatures and high-salt environments. A general explanation of the NaCl effect by means of a statistical thermodynamic model is also provided, together with an analysis of residue distribution between protein surface and interior in 15 non-redundant families of halophilic and non-halophilic proteins. The results are in line with a comparative sequence and structural analysis between halophilic and non-halophilic γ-glutamyltranspeptidases which revealed that a major role in halotolerance should be played by solvent exposed negatively charged residues.

A regular thymine tetrad and a peculiar supramolecular assembly in the first crystal structure of an all-LNA G-quadruplex.

Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2' and C4' atoms are linked through a methylene bridge and the sugar is blocked in a 3'-endo conformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5'-TGGGT-3' is presented refined at 1.7 Šresolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3'-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3'-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.

High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity.

The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human α-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K(+) ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of α-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin-TBA complex formed in the presence of Na(+) or K(+) and a circular dichroism study of the structural stability of the aptamer both free and complexed with α-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na(+) and K(+) on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein-aptamer interface. The present data, in combination with those previously obtained on the complex between α-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.

Structural overview of DNA and RNA G-quadruplexes in their interaction with proteins.

Since the discovery of G-quadruplex (G4) participation in vital cellular processes, the regulation of the interaction of naturally occurring G4s with the relative target proteins has emerged as a promising approach for therapeutic development. Additionally, a synthetic strategy has produced several oligonucleotide aptamers, embodying a G4 module, which exhibit relevant biological activity by binding selectively to a target protein. In this context, the G4-protein structures available in the Protein Data Bank represent a valuable molecular view of the different G4 topologies involved in protein interaction. Interestingly, recent results have shown the co-existence of G4s with other structural domains such as duplexes. Overall, these findings allow a better understanding of the mechanisms that regulate intricate biological functions and suggest new design for innovative medical treatments.

Ordered hierarchical superlattice amplifies coated-CeO2 nanoparticles luminescence.

Achieving a controlled preparation of nanoparticle superstructures with spatially periodic arrangement, also called superlattices, is one of the most intriguing and open questions in soft matter science. The interest in such regular superlattices originates from the potentialities in tailoring the physicochemical properties of the individual constituent nanoparticles, eventually leading to emerging behaviors and/or functionalities that are not exhibited by the initial building blocks. Despite progress, it is currently difficult to obtain such ordered structures; the influence of parameters, such as size, softness, interaction potentials, and entropy, are neither fully understood yet and not sufficiently studied for 3D systems. In this work, we describe the synthesis and characterization of spatially ordered hierarchical structures of coated cerium oxide nanoparticles in water suspension prepared by a bottom-up approach. Covering the CeO2 surface with amphiphilic molecules having chains of appropriate length makes it possible to form ordered structures in which the particles occupy well-defined positions. In the present case superlattice arrangement is accompanied by an improvement in photoluminescence (PL) efficiency, as an increase in PL intensity of the superlattice structure of up to 400 % compared with that of randomly dispersed nanoparticles was observed. To the best of our knowledge, this is one of the first works in the literature in which the coexistence of 3D structures in solution, such as face-centered cubic (FCC) and Frank-Kasper (FK) phases, of semiconductor nanoparticles have been related to their optical properties.

Tailoring the stress response of human skin cells by substantially limiting the nuclear localization of angiogenin.

Human angiogenin (hANG) is the most studied stress-induced ribonuclease (RNase). In physiological conditions it performs its main functions in nucleoli, promoting cell proliferation by rDNA transcription, whereas it is strongly limited by its inhibitor (RNH1) throughout the rest of the cell. In stressed cells hANG dissociates from RNH1 and thickens in the cytoplasm where it manages the translational arrest and the recruitment of stress granules, thanks to its propensity to cleave tRNAs and to induce the release of active halves. Since it exists a clear connection between hANG roles and its intracellular routing, starting from our recent findings on heterologous ANG (ANG) properties in human keratinocytes (HaCaT cells), here we designed a variant unable to translocate into the nucleus with the aim of thoroughly verifying its potentialities under stress. This variant, widely characterized for its structural features and biological attitudes, shows more pronounced aid properties than unmodified protein. The collected evidence thus fully prove that ANG stress-induced skills in assisting cellular homeostasis are strictly due to its cytosolic localization. This study opens an interesting scenario for future studies regarding both the strengthening of skin defences and in understanding the mechanism of action of these special enzymes potentially suitable for any cell type.

On the mechanism of action of arsenoplatins: arsenoplatin-1 binding to a B-DNA dodecamer.

The reaction of Pt-based anticancer agents with arsenic trioxide affords robust complexes known as arsenoplatins. The prototype of this family of anticancer compounds is arsenoplatin-1 (AP-1) that contains an As(OH)2 fragment linked to a Pt(II) moiety derived from cisplatin. Crystallographic and spectrometric studies of AP-1 binding to a B-DNA double helix dodecamer are presented here, in comparison with cisplatin and transplatin. Results reveal that AP-1, cisplatin and transplatin react differently with the DNA model system. Notably, in the AP-1/DNA systems, the Pt-As bond can break down with time and As-containing fragments can be released. These results have implications for the understanding of the mechanism of action of arsenoplatins.