A Crystallographic Approach for Understanding the Recognition Mechanism of Thrombin and G-quadruplex Aptamers

Human-thrombin, a serine protease that maintains blood hemostasis by balancing pro- and anti-coagulant actions is an example of protein with multiple binding sites1. In addition to the active site, the enzyme possesses two electropositive regions, in near-opposition on the protein surface, known as exosite I and exosite II, respectively. These two regions have a primary role in the regulation of enzymatic activity since they can bind molecules with diverse functions2-4. Given its central role in the clot formation, thrombin is an attractive target for the development of agents that effectively interfere with thrombogenesis. A special class of thrombin synthetic ligands is represented by nucleic acid aptamers adopting G-quadruplex structures. HD1, a 15-mer oligonucleotide recognizing exosite I5, and HD22, a 29-mer binding exosite II6, are the most studied thrombin binding aptamers and show high affinity toward their target (Kd (HD1)≈ 100 nM; Kd(HD22) ≈0.7 nM). The increased interest in the use of DNA aptamers as drugs has stimulated the search of HD1 and HD22 variants with improved properties. In particular, the bimodular oligonucleotides RE317 and NU1728, which have been obtained by addition of a duplex motif to the HD1 quadruplex module, show higher affinity for thrombin and anticoagulant activity, and a slower disappearance rate in human plasma in comparison with HD1. Here I will present the most relevant results regarding the elucidation of the interactions, which govern the recognition between thrombin and DNA G-quadruplex aptamers9-14

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 [...]

Monitoring Preparation of Derivative Protein Crystals via Raman Microscopy

This chapter deals with the application of Raman confocal microscopy to monitor the preparation of chemically modified protein crystals. We first introduce the peculiar properties of protein single crystals and related crystallization techniques. Then we move to present experimental sampling and theoretical background of Raman microscopy. Finally we show some examples of applications of Raman microscopy to protein derivative crystal

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

Structural and functional analysis of the simultaneous binding of two duplex/quadruplex aptamers to human α-thrombin

: The long-range communication between the two exosites of human α-thrombin (thrombin) tightly modulates the protein-effector interactions. Duplex/quadruplex aptamers represent an emerging class of very effective binders of thrombin. Among them, NU172 and HD22 aptamers are at the forefront of exosite I and II recognition, respectively. The present study investigates the simultaneous binding of these two aptamers by combining a structural and dynamics approach. The crystal structure of the ternary complex formed by the thrombin with NU172 and HD22_27mer provides a detailed view of the simultaneous binding of these aptamers to the protein, inspiring the design of novel bivalent thrombin inhibitors. The crystal structure represents the starting model for molecular dynamics studies, which point out the cooperation between the binding at the two exosites. In particular, the binding of an aptamer to its exosite reduces the intrinsic flexibility of the other exosite, that preferentially assumes conformations similar to those observed in the bound state, suggesting a predisposition to interact with the other aptamer. This behaviour is reflected in a significant increase of the anticoagulant activity of NU172 when the inactive HD22_27mer is bound to exosite II, providing a clear evidence of the synergic action of the two aptamers

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

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

INFLUENZA DEL SOTTOSUOLO SUL COMPORTAMENTO DINAMICO DEL CAMPANILE DEL CARMINE A NAPOLI

La nota descrive l’identificazione dinamica del campanile del Carmine a Napoli, effettuata attraverso analisi dinamiche su un modello tridimensionale del sistema terreno - fondazione - struttura, tarato sui risultati di dettagliate indagini in situ. Sono stati riconosciuti fenomeni di risonanza tra la frequenza fondamentale del terreno e la seconda frequenza di vibrazione della struttura che ne condizionano il comportamento dinamico

Thrombin–aptamer recognition: a revealed ambiguity

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5′-5′ polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin–mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin–aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs

Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent

A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild-type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N-terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or 68Gallium-labelled synthetic peptide. The labelled peptide permeated MCF-7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with 125I- and 131I-labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro-apoptotic mechanism