Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations

Recently various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. However, the key issue as to the nature of forces driving the folding and recognition processes remains unanswered. In this study, structural changes of 22-mer ht-DNA fragment (Tel22), induced by binding of ions (K⁺, Na⁺) and specific bisquinolinium ligands, were monitored by calorimetric and spectroscopic methods and by gel electrophoresis. Using the global model analysis of a wide variety of experimental data, we were able to characterize the thermodynamic forces that govern the formation of stable Tel22 G-quadruplexes, folding intermediates, and ligand-quadruplex complexes, and then predict Tel22 behavior in aqueous solutions as a function of temperature, salt concentration, and ligand concentration. On the basis of the above, we believe that our work sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism.     

Conformational Transitions upon Ligand Binding: Holo-Structure Prediction from Apo Conformations

Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 Å backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 Å backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.     

X-ray structures of small ligand-FKBP complexes provide an estimate for hydrophobic interaction energies

A new crystal form of native FK506 binding protein (FKBP) has been obtained which has proved useful in ligand binding studies. Three different small molecule ligand complexes and the native enzyme have been determined at higher resolution than 2.0 A. Dissociation constants of the related small molecule ligands vary from 20 mM for dimethylsulphoxide to 200 microM for tetrahydrothiophene 1-oxide. Comparison of the four available crystal structures shows that the protein structures are identical to within experimental error, but there are differences in the water structure in the active site. Analysis of the calculated buried surface areas of these related ligands provides an estimated van der Waals contribution to the binding energy of -0.5 kJ/A(2) for non-polar interactions between ligand and protein.     

On the interaction of an anticancer trisubstituted naphthalene diimide with G-quadruplexes of different topologies: a structural insight

Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology. Interplay of different binding modes of NDI-5 to G-quadruplexes was observed for both parallel and hybrid topologies, with end-stacking always operative as the predominant binding event. While NDI-5 primarily targets the 5′-end quartet of the hybrid G-quadruplex model (m-tel24), the binding to a parallel G-quadruplex model (M2) occurs seemingly simultaneously at the 5′- and 3′-end quartets. With parallel G-quadruplex M2, NDI-5 formed stable complexes with 1:3 DNA:ligand binding stoichiometry. Conversely, when interacting with hybrid G-quadruplex m-tel24, NDI-5 showed multiple binding poses on a single G-quadruplex unit and/or formed different complexes comprising two or more G-quadruplex units. NDI-5 produced stabilizing effects on both G-quadruplexes, forming complexes with dissociation constants in the nM range.     

Interactions of cyclic and non-cyclic naphthalene diimide derivatives with different nucleic acids

Recently, strategy based on stabilization of G-quadruplex telomeric DNA by small organic molecule has been realized by naphthalene diimide derivatives (NDIs). At the same time NDIs bind to DNA duplex as threading intercalators. Here we present cyclic derivative of naphthalene diimide (ligand 1) as DNA-binding ligand with ability to recognition of different structures of telomeric G-quadruplexes and ability to bis-intercalate to double-stranded helixes. The results have been compared to non-cyclic derivative (ligand 2) and revealed that preferential binding of ligands to nucleic acids strongly depends on their topology and structural features of ligands.     

Crystal structures of tRNA-guanine transglycosylase (TGT) in complex with novel and potent inhibitors unravel pronounced induced-fit adaptations and suggest dimer formation upon substrate binding

The bacterial tRNA-guanine transglycosylase (TGT) is a tRNA modifying enzyme catalyzing the exchange of guanine 34 by the modified base preQ1. The enzyme is involved in the infection pathway of Shigella, causing bacterial dysentery. As no crystal structure of the Shigella enzyme is available the homologous Zymomonas mobilis TGT was used for structure-based drug design resulting in new, potent, lin-benzoguanine-based inhibitors. Thorough kinetic studies show size-dependent inhibition of these compounds resulting in either a competitive or non-competitive blocking of the base exchange reaction in the low micromolar range. Four crystal structures of TGT-inhibitor complexes were determined with a resolution of 1.58-2.1 A. These structures give insight into the structural flexibility of TGT necessary to perform catalysis. In three of the structures molecular rearrangements are observed that match with conformational changes also noticed upon tRNA substrate binding. Several water molecules are involved in these rearrangement processes. Two of them demonstrate the structural and catalytic importance of water molecules during TGT base exchange reaction. In the fourth crystal structure the inhibitor unexpectedly interferes with protein contact formation and crystal packing. In all presently known TGT crystal structures the enzyme forms tightly associated homodimers internally related by crystallographic symmetry. Upon binding of the fourth inhibitor the dimer interface, however, becomes partially disordered. This result prompted further analyses to investigate the relevance of dimer formation for the functional protein. Consultation of the available TGT structures and sequences from different species revealed structural and functional conservation across the contacting residues. This suggests that bacterial and eukaryotic TGT could possibly act as homodimers in catalysis. It is hypothesized that one unit of the dimer performs the catalytic reaction whereas the second is required to recognize and properly orient the bound tRNA for the catalytic reaction.     

The Influence of Sequence Microvariation Among HLA-DR3 Alleles on their Structure and Complexes Formation with Presentation Peptides

The three-dimensional structure of human leukocyte antigens HLA-DR*0301 and HLA-DR*0302 have been calculated using the homology modeling approach. General structural features of our models are similar to those of related HLA molecules. The typical layout of segments of the secondary structure is well preserved. However polypeptide chains are less tightly bound, which causes slightly broader opening of the binding groove. It also results in the modified layout of pockets in the binding groove. Amino acids defining the restricted sequence diversity of studied proteins are easily available for interactions with ligands.A set of docking simulations was performed using modeled structures of both HLA molecules and various specific peptide ligands. The control docking of influenza hemagglutinin peptide into HLA-DR*0101 molecule gives the complex structure which is in good agreement with that from crystallographic studies. The extensive analysis of the structure of modeled complexes of HLA-DR*0301 and HLA-DR*0302 with various ligands indicates that sequence microvariation of both alleles is not directly controlling the binding specificity. Preferences for binding of specific ligands, as evaluated from interactions in modeled complexes, agree qualitatively with experimental observations. Thus the computer aided docking simulations can be successfully used to calculate the three-dimensional structure of HLA-ligand complexes. However detailed explanation of binding specificity can not be achieved using presently available modeling procedures.Electronic Supplementary Material available.     

Quadruplex ligands may act as molecular chaperones for tetramolecular quadruplex formation

G-quadruplexes are a family of four-stranded DNA structures, stabilized by G-quartets, that form in the presence of monovalent cations. Efforts are currently being made to identify ligands that selectively bind to G-quadruplex motifs as these compounds may interfere with the telomere structure, telomere elongation/replication and proliferation of cancer cells. The kinetics of quadruplex–ligands interactions are poorly understood: it is not clear whether quadruplex ligands lock into the preformed structure (i.e. increase the lifetime of the structure by lowering the dissociation constant, k_off) or whether ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing the association constant, k_on. We studied the effect of a selective quadruplex ligand, a bisquinolinium pyridine dicarboxamide compound called 360A, to distinguish these two possibilities. We demonstrated that, in addition to binding to and locking into preformed quadruplexes, this molecule acted as a chaperone for tetramolecular complexes by acting on k_on. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when evaluating the cellular responses to these agents.     

Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

We have recently introduced a computational methodology that combines molecular dynamics (MD) simulations, free-energy calculations, and in vitro binding assays to predict the minimum RNA structural requirements for selective, high-affinity RNA binding to small-molecule ligands. Here, we show that this methodology can be applied to the conformationally flexible aminoglycoside antibiotic paromomycin. A RNA consisting of an 11-mer:10-mer duplex that contains one 16S ribosome RNA decoding A-site bound to paromomycin was simulated for 4 ns. The methodology predicts that the 11-mer:10-mer duplex binds to paromomycin with high affinity, whereas smaller RNA duplexes lose complex stability and the ability to bind paromomycin. The predicted high-affinity binding to paromomycin of the 11-mer:10-mer duplex was confirmed experimentally (EC(50) = 0.28 microM), as well as the inability of smaller complexes to bind. Our simulations show good agreement with experiment for dynamic and structural properties of the isolated A-site, including hydrogen-bonding networks and RNA structural rearrangements upon ligand binding. The results suggest that MD simulations can supplement in vitro methods as a tool for predicting minimum RNA-binding motifs for both small, rigid ligands, and large, flexible ligands when structural information is available.     

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.     

Flexible docking using tabu search and an empirical estimate of binding affinity

This article describes the implementation of a new docking approach. The method uses a Tabu search methodology to dock flexibly ligand molecules into rigid receptor structures. It uses an empirical objective function with a small number of physically based terms derived from fitting experimental binding affinities for crystallographic complexes. This means that docking energies produced by the searching algorithm provide direct estimates of the binding affinities of the ligands. The method has been tested on 50 ligand-receptor complexes for which the experimental binding affinity and binding geometry are known. All water molecules are removed from the structures and ligand molecules are minimized in vacuo before docking. The lowest energy geometry produced by the docking protocol is within 1.5 Å root-mean square of the experimental binding mode for 86% of the complexes. The lowest energies produced by the docking are in fair agreement with the known free energies of binding for the ligands. Proteins 33:367–382, 1998. © 1998 Wiley-Liss, Inc.     

Aminoglycoside binding to Oxytricha nova telomeric DNA

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ～ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography.

Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.     

Quadruplex structures in nucleic acids.

DNA oligonucleotides that have repetitive tracts of guanine bases can form G-quadruplex structures that display an amazing polymorphism. Structures of several new G-quadruplexes have been solved recently that greatly expand the known structural motifs observed in nucleic acid quadruplexes. Base triads, base hexads, and quartets that contain cytosine have recently been identified stacked over the familiar G-quartets. The current status of the diverse array of structural features in quadruplexes is described and used to provide insight into the polymorphism and folding pathways. This review also summarizes recent progress in the techniques used to probe the structures of G-quadruplexes and discusses the role of ion binding in quadruplex formation. Several of the quadruplex structures featured in this review can be accessed in the online version of this review as CHIME representations.     

Landscape of protein–small ligand binding modes

Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R² = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.     

48 NMR study of the interaction between G-quadruplexes and small molecules

Guanine-rich oligonucleotides are able to adopt secondary DNA structures, known as G-quadruplexes. Such G-rich sequences are found in human telomeres, promoter regions of oncogenes, 5′ untranslated regions (UTRs) of mRNAs and human intronic sequences. Studies have shown that small molecules can induce anti-cancer effect through stabilizing or promoting G-quadruplex formation. In order to design and develop a potent drug, structural details on the interaction between small molecules and G-quadruplexes are invaluable. In this study, we seek to understand the structural determinants involved in the interaction between G-quadruplexes and small molecules. NMR spectroscopy is employed to resolve the structures of two intramolecular G-quadruplexes bound to small molecules. These resolved complexes allow us to structurally design new potent drugs for their application in anti-cancer therapy.