Curt A. Davey

Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 A Resolution

Solvent binding in the nucleosome core particle containing a 147 base pair, defined-sequence DNA is characterized from the X-ray crystal structure at 1.9 A resolution. A single-base-pair increase in DNA length over that used previously results in substantially improved clarity of the electron density and accuracy for the histone protein and DNA atomic coordinates. The reduced disorder has allowed for the first time extensive modeling of water molecules and ions. Over 3000 water molecules and 18 ions have been identified. Water molecules acting as hydrogen-bond bridges between protein and DNA are approximately equal in number to the direct hydrogen bonds between these components. Bridging water molecules have a dual role in promoting histone-DNA association not only by providing further stability to direct protein-DNA interactions, but also by enabling formation of many additional interactions between more distantly related elements. Water molecules residing in the minor groove play an important role in facilitating insertion of arginine side-chains. Water structure at the interface of the histones and DNA provides a means of accommodating intrinsic DNA conformational variation, thus limiting the sequence dependency of nucleosome positioning while enhancing mobility. Monovalent anions are bound near the N termini of histone alpha-helices that are not occluded by DNA phosphate groups. Their location in proximity to the DNA phosphodiester backbone suggests that they damp the electrostatic interaction between the histone proteins and the DNA. Divalent cations are bound at specific sites in the nucleosome core particle and contribute to histone-histone and histone-DNA interparticle interactions. These interactions may be relevant to nucleosome association in arrays.

The structure of DNA in the nucleosome core

The 1.9-Å-resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein–DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fibre formation and chromatin remodelling.

Ligand substitutions between ruthenium–cymene compounds can control protein versus DNA targeting and anticancer activity

Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favourable toxicity and clearance properties. Nonetheless, their molecular targeting and mechanism of action are poorly understood. Here we study two prototypical ruthenium-arene agents—the cytotoxic antiprimary tumour compound [(η6-p-cymene)Ru(ethylene-diamine)Cl]PF6 and the relatively non-cytotoxic antimetastasis compound [(η6-p-cymene)Ru(1,3,5-triaza-7-phosphaadamantane)Cl2]—and discover that the former targets the DNA of chromatin, while the latter preferentially forms adducts on the histone proteins. Using a novel ‘atom-to-cell’ approach, we establish the basis for the surprisingly site-selective adduct formation behaviour and distinct cellular impact of these two chemically similar anticancer agents, which suggests that the cytotoxic effects arise largely from DNA lesions, whereas the protein adducts may be linked to the other therapeutic activities. Our study shows promise for developing new ruthenium drugs, via ligand-based modulation of DNA versus protein binding and thus cytotoxic potential, to target distinguishing epigenetic features of cancer cells.

A Ruthenium Antimetastasis Agent Forms Specific Histone Protein Adducts in the Nucleosome Core

Keywords: bioinorganic chemistry ; cancer ; histone binding ; nucleosomes ; Rapta-C ; ruthenium ; Mass-Spectrometry ; Anticancer Metallodrugs ; Complexes ; Chromatin ; Binding ; Dna ; Particle ; Chemotherapy ; Selectivity ; Inhibition Reference EPFL-ARTICLE-171621doi:10.1002/chem.201100298View record in Web of Science Record created on 2011-12-16, modified on 2017-05-12

The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association

Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome–nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.

Nucleosome structural studies.

Chromatin plays a fundamental role in eukaryotic genomic regulation, and the increasing awareness of the importance of epigenetic processes in human health and disease emphasizes the need for understanding the structure and function of the nucleosome. Recent advances in chromatin structural studies, including the first structures of nucleosomes containing the Widom 601 sequence and the structure of a chromatin protein-nucleosome assembly, have provided new insight into stretching of nucleosomal DNA, nucleosome positioning, binding of metal ions, drugs and therapeutic candidates to nucleosomes, and nucleosome recognition by nuclear proteins. These discoveries ensure promising future prospects for unravelling structural attributes of chromatin.

DNA-dependent divalent cation binding in the nucleosome core particle

Sequence-specific binding of divalent cations to nucleosomal DNA can potentially influence nucleosome position and mobility, as well as modulate interactions with nuclear factors. We define the bonding and specificity of divalent cation interaction with nucleosomal DNA by characterizing Mn2+ binding in the x-ray structure of the nucleosome core particle at 1.9-Å resolution. Manganese ions are found ligated with high occupancy in the major groove to 12 of 22 GG and GC base pair steps. The specific location and mode of metal binding is the consequence of unambiguous conformational differences between dinucleotide sites, owing to their sequence context and orientation in the nucleosome core.

Novel metal(II) arene 2-pyridinecarbothioamides: a rationale to orally active organometallic anticancer agents

A novel series of organometallic antitumour agents based on RuII and OsII complexes containing N-substituted 2-pyridinecarbothioamides (PCAs) has been synthesized and characterized. To the best of our knowledge, this is the first report of organometallic anticancer compounds with an S,N-bidentate ligand system. While the ligands showed activity as gastric mucosal protectants and low acute toxicity in vivo (J. Med. Chem., 1990, 33, 327–336), coordination leads to highly antiproliferative metallodrugs, depending on lipophilicity and steric demand, in colon carcinoma and non-small lung cancer cell lines with intrinsic chemoresistances. The most lipophilic and smallest congeners are the most effective with IC50 values in the low micromolar range. This new family of potential metallodrugs features exceptional stability in hydrochloric acid (60 mM), characterized by complete suppression of hydrolysis and low reactivity towards biological nucleophiles. Therefore, their unexpected aqueous chemistry renders this family of antiproliferative agents suitable for oral administration. An unprecedented feature is their ability to form transient thioketone-bridged dimers in aqueous solution upon hydrolysis, which is believed to minimize deactivation by biological nucleophiles. However, the biological effect seems to be caused by the monomer as observed with crystallographic studies of the nucleosome core particle (NCP), which revealed that [chlorido(η6-p-cymene)(N-phenyl-2-pyridinecarbothioamide)osmium(II)] chloride and [chlorido(η6-p-cymene)(N-fluorophenyl-2-pyridinecarbothioamide)osmium(II)] chloride react at two types of binding sites on the histone proteins. The adducts form at histidine side chains located on the nucleosome surface and the inner cleft of the nucleosome in the midst of an extensive histone–histone interface, suggesting interference with chromatin activity as a possible mode of action of these compounds. Additionally, ligand-based S → O exchange allows for a potential dual-mode of action by targeting DNA (J. Med. Chem., 2009, 52, 7753–7764). The quantitative estimates of drug-likeness (QED) for this family of compounds revealed a similar drug-likeness compared to erlotinib, tamoxifen, imatinib and sorafenib.

Crystal structure of Escherichia coli alkanesulfonate monooxygenase SsuD.

The FMNH(2)-dependent alkanesulfonate monooxygenase SsuD catalyzes the conversion of alkanesulfonates to the corresponding aldehyde and sulfite. The enzyme allows Escherichia coli to use a wide range of alkanesulfonates as sulfur sources for growth when sulfate or cysteine are not available. The structure of SsuD was solved using the multiwavelength anomalous dispersion method from only four ordered selenium sites per asymmetric unit (one site per 20,800 Da). The final model includes 328 of 380 amino acid residues and was refined to an R-factor of 23.5% (R(free)=27.5%) at 2.3A resolution. The X-ray crystal structure of SsuD shows a homotetrameric state for the enzyme, each subunit being composed of a TIM-barrel fold enlarged by four insertion regions that contribute to intersubunit interactions. SsuD is structurally related to a bacterial luciferase and an archaeal coenzyme F(420)-dependent reductase in spite of a low level of sequence identity with these enzymes. The structural relationship is not limited to the beta-barrel region; it includes most but not all extension regions and shows distinct properties for the SsuD TIM-barrel. A likely substrate-binding site is postulated on the basis of the SsuD structure presented here, results from earlier biochemical studies, and structure relatedness to bacterial luciferase. SsuD is related to other FMNH(2)-dependent monooxygenases that show distant sequence relationship to luciferase. Thus, the structure reported here provides a model for enzymes belonging to this family and suggests that they might all fold as TIM-barrel proteins.

Fighting Cancer with Transition Metal Complexes: From Naked DNA to Protein and Chromatin Targeting Strategies

Many transition metal complexes have unique physicochemical properties that can be efficiently exploited in medicinal chemistry for cancer treatment. Traditionally, double‐stranded DNA has been assumed to be the main binding target; however, recent studies have shown that nucleosomal DNA as well as proteins can act as dominant molecular binding partners. This has raised new questions about the molecular determinants that govern DNA versus protein binding selectivity, and has offered new ways to rationalize their biological activity and possible side effects. To address these questions, molecular simulations at an atomistic level of detail have been used to complement, support, and rationalize experimental data. Herein we review some relevant studies—focused on platinum and ruthenium compounds—to illustrate the power of state‐of‐the‐art molecular simulation techniques and to demonstrate how the interplay between molecular simulations and experiments can make important contributions to elucidating the target preferences of some promising transition metal anticancer agents. This contribution aims at providing relevant information that may help in the rational design of novel drug‐discovery strategies.