Disorders of the blood coagulation cascade continue to pose a major clinical challenge, necessitating the development of new therapeutic agents capable of modulating this process. Several oligonucleotide aptamers targeting coagulation factors have been developed, and some are undergoing preclinical or clinical evaluation. Among them, anti-thrombin anticoagulant aptamers are promising dual-targeting agents in that, in addition to inhibiting enzyme activity, they may limit thrombin generation by binding to its precursor, prothrombin. In the present study, combined calorimetric and spectroscopic analyses reveal that these aptamers recognize proexosite I of prothrombin and exosite I of thrombin through broadly similar thermodynamic binding mechanisms. Integration of structural small-angle X-ray scattering (SAXS) studies and limited proteolysis assays shows that aptamer binding to proexosite I alters prothrombin structure, shifting the equilibrium from its more abundant closed form to the open conformation. Taken together, these results support the classification of these aptamers as dual-targeting agents capable of recognizing both thrombin and prothrombin and provide guidance for their continued development as anticoagulant therapeutics.

The molecular mechanisms by which the human CDC25B activates the CDK1/cyclin B complex in the cell cycle, as well as how it can be inhibited by synthetic inhibitors at the atomic level, are still under investigation. Valuable insights have been gained from the molecular structure here-described, which captures for the first time the interaction between the C-terminal domain of the inactive mutant CDC25B C473S (CDC25B-S) and the commonly used synthetic substrate 3-O-methylfluorescein phosphate (3-OMFP). Crystallographic studies reveal that 3-OMFP engages multiple residues within the active site and the adjacent "swimming pool" of CDC25B-S, establishing specific interactions and prompting local adjustments in this region. These structural features explain the increased resistance to thermal denaturation of CDC25B-S observed through circular dichroism measurements upon substrate binding. The structural changes induced by 3-OMFP lead to a conformation comparable to that of CDC25A bound to its substrate, the CDK2/cyclin A complex. These findings qualify 3-OMFP as a promising starting model for the rational design of selective competitive inhibitors of CDC25B having reduced off-target effects.

The interaction of aurothiomalate with the apo-form of transferrin (hTF) was studied by X-ray crystallography. Data provide information on gold binding sites and on a unique structure along the apo-hTF/holo-hTF transition pathway.

Disorders of the blood coagulation remain a leading cause of death and disability worldwide raising the search for therapeutic agents able to modulate the coagulation cascade. Different oligonucleotide aptamers have been selected against different coagulation factors and some of them are in preclinical or clinical studies. In particular, anti-thrombin aptamers are promising drugs as they inhibit the activity of the α-thrombin and, simultaneously, limit thrombin production via prothrombinase by binding its precursor prothrombin. To investigate the interaction of these aptamers with prothrombin, we performed extensive analyses using calorimetric and spectroscopic techniques, which suggested that they recognize proexosite I of prothrombin and exosite I of thrombin with comparable affinity. SAXS experiments performed on the complex formed by the protein and NU172, the only anti-thrombin aptamer in advanced clinical trials, provided structural insights into aptamer-prothrombin recognition. Interestingly, the aptamer binding to proexosite I shifts the open-closed equilibrium of prothrombin toward the open conformation. A reasonable mechanism underlying the effects of anti-thrombin aptamers towards prothrombin conversion into thrombin has been proposed. Altogether, these results definitively qualify these aptamers as bitargeted drugs, being able to modulate both thrombin function and generation, and supply structural bases to design new anticoagulants, which lack health side effects. ### Competing Interest Statement The authors have declared no competing interest.

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 FrankKasper (FK) phases, of semiconductor nanoparticles have been related to their optical properties.

Crystallographic and spectrometric studies of AP-1, cisplatin and transplatin binding to a B-DNA double helix dodecamer are reported.

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.

Here, we report the X-ray structure of the adduct formed upon reaction of cisplatin, one of the most prescribed anticancer agents for the clinic treatment of solid tumors, with the apo-form of human serum transferrin (hTF). Two Pt binding sites were identified in both molecules of the adduct present in the crystal asymmetric unit: Pt binds close to the side chains of Met256 and Met499 at the N- and C-lobe, respectively. In the crystal structure, the cisplatin moiety bound to Met256 also interacts with Ser616 from a symmetry related molecule. Structural analyses, together with in solution data, demonstrate that the presence of iron does not affect the ability of hTF to bind cisplatin and that the cisplatin binding does not significantly alter the overall conformation of the different forms of the protein that remain able to form a complex with the transferrin receptor 1 (TfR1). These data suggest that the different hTF forms can be used as nanocarriers for targeted (combined) metallodrug delivery.

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.

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

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.

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.

The coagulation process relies on an intricate network of three-dimensional structural interactions and subtle biological regulations. In the present review, we illustrate the state of the art of the structural biology of the coagulation cascade by surveying the Protein Data Bank and the EBI AlphaFold databases. Investigations performed in the last decade have provided structural information on essentially all players involved in the process. Indeed, the initial characterization of specific and rather canonical domains has been progressively extended to complicated multidomain proteins. Recently, the application of cryogenic electron microscopy techniques has unraveled the structural features of highly complex coagulation factors, which has led to enhanced understanding. This review initially focuses on the structure of the individual factors as a function of their involvement in intrinsic, extrinsic, and common pathways. A specific emphasis is given to what is known or unknown on the structural basis of each step of the cascade. Available data providing clues on the structural recognition of the factors involved in the functional partnerships of the pathways are illustrated. Recent structures of important complexes formed by these proteins with regulators are described, focusing on the drugs used as anticoagulants and on their reversal agents. Finally, we highlight the different roles that innovative biomolecules such as aptamers may have in the regulation of the cascade.

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.

The reaction of the cytotoxic compound dirhodium tetraacetate with a B-DNA double helical dodecamer was studied by X-ray crystallography and mass spectrometry. The structure of the dirhodium/DNA adduct reveals a dimetallic center binding to an adenine via axial coordination. Complementary information has been gained through ESI MS measurements. Comparison between the present data and those previously obtained for cisplatin indicates that the two metallodrugs react with this DNA dodecamer in a significantly different fashion.

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.

Despite their unquestionable properties, oligonucleotide ap-tamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to over-come these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moi-eties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 50 and 30 ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallo-graphic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/ DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel abil-ity is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a pseudo-cyclic charge -transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities.

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.

The superfamily of vertebrate ribonucleases, a large group of evolutionarily related proteins, continues to provide interesting structural and functional information. In particular, the crystal structure of SS-RNase-2 from Salmo salar (SS2), here presented, has revealed a novel auto-inhibition mechanism that enriches the number of inhibition strategies observed in some members of the family. Within an essentially unmodified RNase folding, the SS2 active site cleft is in part obstructed by the collapse of an extra pentapeptide inserted in the C-terminal region. This unexpected intrusion alters the organization of the catalytic triad by pushing one catalytic histidine off the pocket. Possible mechanisms to remove the active site obstruction have also been studied through the production of two mutants that provide useful information on the functionality of this intriguing version of the ribonuclease superfamily.

Human α-thrombin (thrombin) is a multifunctional enzyme that plays a pivotal role in the coagulation pathway. Thrombin activity can be effectively modulated by G-quadruplex-based oligonucleotide aptamers that specifically interact with the two positively charged regions (exosites I and II) on the protein surface. Although insightful atomic-level snapshots of the recognition between thrombin and aptamers have been recently achieved through crystallographic analyses, some dynamic aspects of this interaction have not been fully characterized. We here report molecular dynamics simulations of thrombin in different association states: ligand-free and binary/ternary complexes with the aptamers TBA (at exosite I) and HD22_27mer (at exosite II). The simulations carried out on the binary and ternary complexes formed by thrombin with these aptamers provide a dynamic view of the interactions that stabilize them in a crystal-free environment. Interestingly, the analysis of the dynamics of the exosites in different thrombin binding states clearly indicates that the HD22_27mer binding at the exosite II favours conformations of exosite I that are prone to the TBA binding. Similar effects are observed upon the binding of TBA to the exosite I. These observations provide an atomic-level picture of the exosite inter-communication in thrombin and explain the experimentally detected cooperativity of the TBA/HD22_27mer binding.