Role of stacking interactions in the binding sequence preferences of DNA bis-intercalators: insight from thermodynamic integration free energy simulations

The major structural determinant of the preference to bind to CpG binding sites on DNA exhibited by the natural quinoxaline bis-intercalators echinomycin and triostin A, or the quinoline echinomycin derivative, 2QN, is the 2-amino group of guanine (G). However, relocation of this group by means of introduction into the DNA molecule of the 2-aminoadenine (=2,6-diaminopurine, D) base in place of adenine (A) has been shown to lead to a drastic redistribution of binding sites, together with ultratight binding of 2QN to the sequence DTDT. Also, the demethylated triostin analogs, TANDEM and CysMeTANDEM, which bind with high affinity to TpA steps in natural DNA, bind much less tightly to CpI steps, despite the fact that both adenosine and the hypoxanthine-containing nucleoside, inosine (I), provide the same hydrogen bonding possibilities in the minor groove. To study both the increased binding affinity of 2QN for DTDT relative to GCGC sites and the remarkable loss of binding energy between CysMeTANDEM and ICIC compared with ATAT, a series of thermodynamic integration free energy simulations involving conversions between DNA base pairs have been performed. Our results demonstrate that the electrostatic component of the stacking interactions between the heteroaromatic rings of these compounds and the bases that make up the intercalation sites plays a very important role in the modulation of their binding affinities

Role of stacking interactions in the binding sequence preferences of DNA bis-intercalators: insight from thermodynamic integration free energy simulations

The major structural determinant of the preference to bind to CpG binding sites on DNA exhibited by the natural quinoxaline bis-intercalators echinomycin and triostin A, or the quinoline echinomycin derivative, 2QN, is the 2-amino group of guanine (G). However, relocation of this group by means of introduction into the DNA molecule of the 2-aminoadenine (=2,6-diaminopurine, D) base in place of adenine (A) has been shown to lead to a drastic redistribution of binding sites, together with ultratight binding of 2QN to the sequence DTDT. Also, the demethylated triostin analogs, TANDEM and CysMeTANDEM, which bind with high affinity to TpA steps in natural DNA, bind much less tightly to CpI steps, despite the fact that both adenosine and the hypoxanthine-containing nucleoside, inosine (I), provide the same hydrogen bonding possibilities in the minor groove. To study both the increased binding affinity of 2QN for DTDT relative to GCGC sites and the remarkable loss of binding energy between CysMeTANDEM and ICIC compared with ATAT, a series of thermodynamic integration free energy simulations involving conversions between DNA base pairs have been performed. Our results demonstrate that the electrostatic component of the stacking interactions between the heteroaromatic rings of these compounds and the bases that make up the intercalation sites plays a very important role in the modulation of their binding affinities

Solution structure and stability of a disulfide cross-linked nucleopeptide duplex

NMR methods are used to study the structure and stability of the duplex formed by the nucleopeptide [Ac- Cys- Gly- Ala-Hse( p(3')dGCATGC)- Ala-OH](2)[S-S], in which the oligonucleotide is self-complementary and the cysteine residues of the two peptide chains form a disulfide bridge; thermal transitions and NMR-derived structural calculations are consistent with a 3-D structure in which the oligonucleotide forms a standard B-DNA helix without significant distortions; the peptide chains are relatively disordered in solution and lie in the minor groove of the DNA helix; this nucleopeptide duplex exhibits a high melting temperature, indicating that peptide - oligonucleotide conjugates containing cysteines are suitable molecules to establish cross-links between DNA strands and stabilize the duplex

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate.

ABSTRACT Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluR

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate.

ABSTRACT Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluR

Stepwise dissection and visualization of the catalytic mechanism of haloalkane dehalogenase LinB using molecular dynamics simulations and computer graphics

Abstract The different steps of the dehalogenation reaction carried out by LinB on three different substrates have been characterized using a combination of quantum mechanical calculations and molecular dynamics simulations. This has allowed us to obtain information in atomic detail about each step of the reaction mechanism, that is, substrate entrance and achievement of the near-attack conformation, transition state stabilization within the active site, halide stabilization, water molecule activation and subsequent hydrolytic attack on the ester intermediate with formation of alcohol, and finally product release. Importantly, no bias or external forces were applied during the whole procedure so that both intermediates and products were completely free to sample configuration space in order to adapt to the plasticity of the active site and/or search for an exit. Differences in substrate reactivity were found to be correlated with the ease of adopting the near-attack conformation and two different exit pathways were found for product release that do not interfere with substrate entrance. Additional support for the different entry and exit pathways was independently obtained from an examination of the enzyme's normal modes.

Dimerization inhibitors of HIV-1 reverse transcriptase, protease and integrase: A single mode of inhibition for the three HIV enzymes?

The genome of human immunodeficiency virus type 1 (HIV-1) encodes 15 distinct proteins, three of which provide essential enzymatic functions: a reverse transcriptase (RT), an integrase (IN), and a protease (PR). Since these enzymes are all homodimers, pseudohomodimers or multimers, disruption of protein-protein interactions in these retroviral enzymes may constitute an alternative way to achieve HIV-1 inhibition. A growing number. of dimerization inhibitors for these enzymes is being reported. This mini review summarizes some approaches that have been followed for the development of compounds that inhibit those three enzymes by interfering with the dimerization interfaces between the enzyme subunits. (c) 2006 Elsevier B.V. All rights reserved

Las glauconitas de la plataforma continental gallega: indicadores geoquímicos del grado de evolución.

La interpretación estratigráfica de unidades que contienen glauconita requiere de una adecuada caracterización del grado de evolución, definido a partir del concepto de madurez (Amorosi, 1995). Su utilización es útil para determinar la duración de periodos asociados a bajas tasas de sedimentación o para caracterizar condiciones paleoceanográficas y paleoambientales en la interfase sedimento-agua del mar. En este tipo de estudios se asume implícitamente que la glauconita actúa como un sistema abierto, susceptible de continuos cambios en la estequiometría, como resultado del intercambio de cationes con el agua del mar que, a su vez, inducen reajustes estructurales. La determinación del grado de evolución de glauconitas está asociada habitualmente a la presencia de determinados marcadores, algunos de tipo cualitativo como es el color o bien asociados a la variación de alguna de sus propiedades físicas, como es la susceptibilidad magnética o composicionales y estructurales, como son la determinación del contenido en K+ o la distancia entre las reflexiones 001, 020 en diagramas de difracción de rayos X. Sin embargo, una caracterización adecuada del proceso evolutivo en glauconita que pueda ser aplicada de forma general debe ser capaz de establecer una relación precisa entre los cambios químicos con el medio durante el proceso de creci 233 S. Fernández-Bastero et al. Las glauconitas de la plataforma continental gallega... miento y la tendencia hacia una mayor estabilidad termodinámica, conectada con modificaciones estructurales, desde una etapa inicial de formación, casi amorfa, hasta alcanzar los últimos términos de la solución sólida, más ordenados. En este sentido, este trabajo se ha planteado con una doble finalidad: en una primera etapa se ha determinado la variabilidad composicional en glauconitas de la plataforma continental gallega y en segundo lugar, estos valores han sido comparados con la variabilidad estructural al objeto de establecer patrones de comportamiento durante las diferentes etapas del proceso evolutivo. El desarrollo de este estudio está basado en el análisis cuantitativo de los diagramas de difracción de rayos X de 50 muestras de sedimento superficial, mediante el método de Rietveld. Con este fin, se ha desarrollado una aplicación particular de este procedimiento de análisis, basada en el ajuste simultáneo de los factores de ocupación para las distintas sustituciones en lugares tetraédricos y octaédricos, así como en las intercapas de la estructura. De este modo, las modificaciones en la estequiometría, inducidas por los procesos de intercambio catiónico entre la superficie mineral y el agua del mar se pueden relacionar con cambios estructurales asociados a las diferentes etapas del proceso evolutivo. Como resultado de este análisis, ha sido posible la elaboración de mapas de contenido geoquímico que reflejan la distribución espacial, tanto para la variabilidad composicional como para la cristalinidad en las glauconitas asociadas a sedimentos superficiales de la plataforma continental gallega

Mechanistic insight into the reaction catalysed by bacterial type II dehydroquinases

DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymesFinancial support from the Comunidad de Madrid (S2010-BMD-2457 to F.G.), Xunta de Galicia (10PXIB2200122PR and GRC2010/12 to C.G.-B.) and the Spanish Ministry of Science and Innovation (SAF2009-13914-C02-02 to F.G. and SAF2010-15076 to C.G.-B.) is 5076 to CGB and BFU2008-01588/BMC to MJvR) is gratefully acknowledged. C.C. and A.P. thank the Spanish Ministry of Science and Innovation for their respective FPU fellowshipsS

XPF-Dependent DNA Breaks and RNA Polymerase II Arrest Induced by Antitumor DNA Interstrand Crosslinking-Mimetic Alkaloids

SummaryTrabectedin and Zalypsis are two potent anticancer tetrahydroisoquinoline alkaloids that can form a covalent bond with the amino group of a guanine in selected triplets of DNA duplexes and eventually give rise to double-strand breaks. Using well-defined in vitro and in vivo assays, we show that the resulting DNA adducts stimulate, in a concentration-dependent manner, cleavage by the XPF/ERCC1 nuclease on the strand opposite to that bonded by the drug. They also inhibit RNA synthesis by: (1) preventing binding of transcription factors like Sp1 to DNA, and (2) arresting elongating RNA polymerase II at the same nucleotide position regardless of the strand they are located on. Structural models provide a rationale for these findings and highlight the similarity between this type of DNA modification and an interstrand crosslink