Global Interactome Mapping Reveals Pro-tumorigenic Interactions of NF-κB in Breast Cancer.

NF-κB pathway is involved in inflammation; however, recent data shows its role also in cancer development and progression, including metastasis. To understand the role of NF-κB interactome dynamics in cancer, we study the complexity of breast cancer interactome in luminal A breast cancer model and its rearrangement associated with NF-κB modulation. Liquid chromatography-mass spectrometry measurement of 160 size-exclusion chromatography fractions identifies 5460 protein groups. Seven thousand five hundred sixty eight interactions among these proteins have been reconstructed by PrInCE algorithm, of which 2564 have been validated in independent datasets. NF-κB modulation leads to rearrangement of protein complexes involved in NF-κB signaling and immune response, cell cycle regulation, and DNA replication. Central NF-κB transcription regulator RELA co-elutes with interactors of NF-κB activator PRMT5, and these complexes are confirmed by AlphaPulldown prediction. A complementary immunoprecipitation experiment recapitulates RELA interactions with other NF-κB factors, associating NF-κB inhibition with lower binding of NF-κB activators to RELA. This study describes a network of pro-tumorigenic protein interactions and their rearrangement upon NF-κB inhibition with potential therapeutic implications in tumors with high NF-κB activity.

Importance of base-pair opening for mismatch recognition.

Mismatch repair is a highly conserved cellular pathway responsible for repairing mismatched dsDNA. Errors are detected by the MutS enzyme, which most likely senses altered mechanical property of damaged dsDNA rather than a specific molecular pattern. While the curved shape of dsDNA in crystallographic MutS/DNA structures suggests the role of DNA bending, the theoretical support is not fully convincing. Here, we present a computational study focused on a base-pair opening into the minor groove, a specific base-pair motion observed upon interaction with MutS. Propensities for the opening were evaluated in terms of two base-pair parameters: Opening and Shear. We tested all possible base pairs in anti/anti, anti/syn and syn/anti orientations and found clear discrimination between mismatches and canonical base-pairs only for the opening into the minor groove. Besides, the discrimination gap was also confirmed in hotspot and coldspot sequences, indicating that the opening could play a more significant role in the mismatch recognition than previously recognized. Our findings can be helpful for a better understanding of sequence-dependent mutability. Further, detailed structural characterization of mismatches can serve for designing anti-cancer drugs targeting mismatched base pairs.

Modes of Micromolar Host-Guest Binding of ß-Cyclodextrin Complexes Revealed by NMR Spectroscopy in Salt Water.

Multitopic supramolecular guests with finely tuned affinities toward widely explored cucurbit[n]urils (CBs) and cyclodextrins (CDs) have been recently designed and tested as functional components of advanced supramolecular systems. We employed various spacers between the adamantane cage and a cationic moiety as a tool for tuning the binding strength toward CB7 to prepare a set of model guests with KCB7 and Kß-CD values of (0.6-5.0) × 1010 M-1 and (0.6-2.6) × 106 M-1, respectively. These accessible adamantylphenyl-based binding motifs open a way toward supramolecular components with an outstanding affinity toward ß-cyclodextrin. 1H NMR experiments performed in 30% CaCl2/D2O at 273 K along with molecular dynamics simulations allowed us to identify two arrangements of the guest@ß-CD complexes. The approach, joining experimental and theoretical methods, provided a better understanding of the structure of cyclodextrin complexes and related molecular recognition, which is highly important for the rational design of drug delivery systems, molecular sensors and switches.

Bending of Canonical and G/T Mismatched DNAs.

Mismatched base pairs alter the flexibility and intrinsic curvature of DNA. The role of such DNA features is not fully understood in the mismatch repair pathway. MutS/DNA complexes exhibit DNA bending, PHE intercalation, and changes of base-pair parameters near the mismatch. Recently, we have shown that base-pair opening in the absence of MutS can discriminate mismatches from canonical base pairs better than DNA bending. However, DNA bending in the absence of MutS was found to be rather challenging to describe correctly. Here, we present a computational study on the DNA bending of canonical and G/T mismatched DNAs. Five types of geometric parameters covering template-based bending toward the experimental DNA structure, global, and local geometry parameters were employed in biased molecular dynamics in the absence of MutS. None of these parameters showed higher discrimination than the base-pair opening. Only roll could induce a sharply localized bending of DNA as observed in the experimental MutS/DNA structure. Further, we demonstrated that the intercalation of benzene mimicking PHE decreases the energetic cost of DNA bending without any effect on mismatch discrimination.

A Photochemical/Thermal Switch Based on 4,4'-Bis(benzimidazolio)stilbene: Synthesis and Supramolecular Properties.

Stilbene derivatives are well-recognised substructures of molecular switches based on photochemically and/or thermally induced (E)/(Z) isomerisation. We combined a stilbene motif with two benzimidazolium arms to prepare new sorts of supramolecular building blocks and examined their binding properties towards cucurbit[n]urils (n=7, 8) and cyclodextrins (ß-CD, γ-CD) in water. Based on the 1 H NMR data and molecular dynamics simulations, we found that two distinct complexes with different stoichiometry, i. e., guest@ß-CD and guest@ß-CD2 , coexist in equilibrium in a water solution of the (Z)-stilbene-based guests. We also demonstrated that the bis(benzimidazolio)stilbene guests can be transformed from the (E) into the (Z) form via UV irradiation and back via thermal treatment in DMSO.

Conformationally Mobile Acyclic Cucurbit[n]uril-Type Receptors Derived from an S-shaped Methylene Bridged Glycoluril Pentamer.

We report the synthesis of the conformationally mobile S-shaped glycoluril pentamer building block 3a and two new acyclic CB[n]-type receptors P1 and P2. P1 (9 mM) and P2 (11 mM) have moderate aqueous solubility but their host•guest complexes are poorly soluble. Host P1 does not undergo intermolecular self-association whereas P2 does (Ks = 189±27 M-1). 1H NMR titrations show that P1 and P2 are poor hosts toward hydrophobic (di)cations 6 - 11 (P1: Ka = 375-1400 M-1; P2: Ka = 1950-19800 M-1) compared to Tet1 and Tet2 (Tet1: Ka = 3.09 × 106 to 4.69 × 108 M-1; Tet2: Ka = 4.59 × 108 to 1.30 × 1010 M-1). Molecular modelling shows that P1 and P2 exist as a mixture of three different conformers due to the two S-shaped methylene bridged glycoluril dimer subunits that each possess two different conformations. The lowest energy conformers of P1 and P2 do not feature a well-defined central cavity. In the presence of guests, P2 adapts its conformation to form 1:1 P2•guest complexes; the binding free energy pays the energetic price of conformer selection. This energetically unfavorable conformer selection results in significantly decreased Ka values of P1 and P2 compared to Tet1 and Tet2.

Bambusuril as a One-Electron Donor for Photoinduced Electron Transfer to Methyl Viologen in Mixed Crystals.

Methyl viologen hexafluorophosphate (MV2+·2PF6-) and dodecamethylbambus[6]uril (BU6) form crystals in which the layers of viologen dications alternate with those of a 1:2 supramolecular complex of BU6 and PF6-. This arrangement allows for a one-electron reduction of MV2+ ions upon UV irradiation to form MV+• radical cations within the crystal structure with half-lives of several hours in air. The mechanism of this photoinduced electron transfer in the solid state and the origin of the long-lived charge-separated state were studied by steady-state and transient spectroscopies, cyclic voltammetry, and electron paramagnetic resonance spectroscopy. Our experiments are supported by quantum-chemical calculations showing that BU6 acts as a reductant. In addition, analogous photochemical behavior is also demonstrated on other MV2+/BU6 crystals containing either BF4- or Br- counterions.

A Cucurbituril Derivative That Exhibits Cation-Modulated Self-Assembly.

Cucurbiturils are the most potent artificial receptors known for many organic molecules in water. However, little is known about their supramolecular chemistry in organic solvents. Here we present a new cucurbituril derivative, 1, and investigate its supramolecular properties in methanol. The macrocycle resembles a five-membered cucurbituril in which four glycoluril units are replaced with propanediurea. Macrocycle 1 can bind to one cation such as potassium or anilinium via each of its opposed portals. The stability of these complexes in methanol at nanomolar concentrations exceeds that of complexes between metal cations and crown ethers. Moreover, macrocycle 1 forms a self-assembled tetrameric aggregate in the solid state and in methanol. The tetramer is stabilized by the addition of up to 1 equiv of a cation but is fully disassembled in the presence of 2 equiv of the cation. Cations can thus be used to tune the aggregation of 1 in solution.

Cooperative Binding of Cucurbit[n]urils and ß-Cyclodextrin to Heteroditopic Imidazolium-Based Guests.

Imidazolium-based guests containing two distinct binding epitopes are capable of binding ß-cyclodextrin and cucurbit[6/7]uril (CB) simultaneously to form heteroternary 1:1:1 inclusion complexes. In the final configuration, the hosts occupy binding sites disfavored in the binary complexes because of the chemically induced reorganization of the intermediate 1:1 aggregate. In addition, the reported guests are capable of binding two CBs to form either 1:2 or 1:1:1 ternary assemblies despite consisting of a single cationic moiety. Whereas the adamantane site binds CB solely via hydrophobic interactions, the CB unit at the butyl site is stabilized by a combination of hydrophobic and ion-dipole interactions.

Tailoring the properties of quadruplex nucleobases for biological and nanomaterial applications.

Guanine DNA quadruplexes are interesting and important biological objects because they represent potential targets for regulatory drugs. Their use as building blocks for biomaterial applications is also being investigated. This contribution reports the in silico design of artificial building blocks derived from xanthine. Methods of quantum chemistry were used to evaluate the properties of xanthine structures relative to those containing guanine, the natural reference used. Tailoring the xanthine core showed that the base stacking and the ion coordination were significantly enhanced in the designed systems, while the ion-transport properties were not affected. Our study suggests that the 9-deaza-8-haloxanthine bases (where the halogen is fluorine or chlorine) are highly promising candidates for the development of artificial quadruplexes and quadruplex-active ligands.

129Xe NMR chemical shift in Xe@C60 calculated at experimental conditions: essential role of the relativity, dynamics, and explicit solvent.

The isotropic (129)Xe nuclear magnetic resonance (NMR) chemical shift (CS) in Xe@C60 dissolved in liquid benzene was calculated by piecewise approximation to faithfully simulate the experimental conditions and to evaluate the role of different physical factors influencing the (129)Xe NMR CS. The (129)Xe shielding constant was obtained by averaging the (129)Xe nuclear magnetic shieldings calculated for snapshots obtained from the molecular dynamics trajectory of the Xe@C60 system embedded in a periodic box of benzene molecules. Relativistic corrections were added at the Breit-Pauli perturbation theory (BPPT) level, included the solvent, and were dynamically averaged. It is demonstrated that the contribution of internal dynamics of the Xe@C60 system represents about 8% of the total nonrelativistic NMR CS, whereas the effects of dynamical solvent add another 8%. The dynamically averaged relativistic effects contribute by 9% to the total calculated (129)Xe NMR CS. The final theoretical value of 172.7 ppm corresponds well to the experimental (129)Xe CS of 179.2 ppm and lies within the estimated errors of the model. The presented computational protocol serves as a prototype for calculations of (129)Xe NMR parameters in different Xe atom guest-host systems.

Determination of intrinsic binding modes by mass spectrometry: gas-phase behavior of adamantylated bisimidazolium guests complexed to cucurbiturils.

Adamantylated bisimidazolium cations exhibit a distinct fragmentation pathway in contrast to their cucurbit[7]uril (CB7) complexes. The observed alternative fragmentation of the guest molecule in a complex clearly correlates to the supposed sterically hindered or allowed slippage of the macrocycle over the axel molecule.

Electron density shift in imidazolium derivatives upon complexation with cucurbit[6]uril.

In this study, we have investigated the supramolecular interaction between series of 1-alkyl-3-methylimidazolium guests with variable alkyl substituent lengths and cucurbit[6]uril (CB6) in the solution and the solid state. Correct interpretation of (1)H NMR spectra was a key issue for determining the binding modes of the complexes in solution. Unusual chemical shifts of some protons in the (1)H NMR spectra were explained by the polarization of the imidazolium aromatic ring upon the complexation with the host. The formation of 1:1 complex between 1-ethyl-3-methylimidazolium and CB6 is in disagreement with previously reported findings describing an inclusion of two guest molecules in the CB6 cavity.

The energy gap as a universal reaction coordinate for the simulation of chemical reactions.

The selection of a proper reaction coordinate is a major bottleneck in simulations of chemical reactions in complex systems. Increasing the number of variables that are used to bias the reaction largely affects the convergence and leads to an unbearable increase in computational price. This problem can be overcome by employing a complex reaction coordinate that depends on many geometrical variables of the system, such as the energy gap (EGAP) in the empirical valence bond (EVB) method. EGAP depends on all of the coordinates of the system, and its robustness has been demonstrated for a variety of enzymatic reactions. In this work, we demonstrate that EGAP, derived from a classical representation, can be used as a reaction coordinate in systems described with any quantum chemistry Hamiltonian. Benefits of using EGAP as a reaction coordinate as compared to a traditional geometrical variable are illustrated in the case of a symmetric nucleophilic substitution reaction in water solution. EGAP is shown to provide a significantly more efficient sampling and allows a better localization of the transition state as compared to a geometrical reaction coordinate.

Evaluating boundary dependent errors in QM/MM simulations.

Hybrid quantum mechanics/molecular mechanics (QM/MM) simulations provide a powerful tool for studying chemical reactions, especially in complex biochemical systems. In most works to date, the quantum region is kept fixed throughout the simulation and is defined in an ad hoc way based on chemical intuition and available computational resources. The simulation errors associated with a given choice of the quantum region are, however, rarely assessed in a systematic manner. Here we study the dependence of two relevant quantities on the QM region size: the force error at the center of the QM region and the free energy of a proton transfer reaction. Taking lysozyme as our model system, we find that in an apolar region the average force error rapidly decreases with increasing QM region size. In contrast, the average force error at the polar active site is considerably higher, exhibits large oscillations and decreases more slowly, and may not fall below acceptable limits even for a quantum region radius of 9.0 A. Although computation of free energies could only be afforded until 6.0 A, results were found to change considerably within these limits. These errors demonstrate that the results of QM/MM calculations are heavily affected by the definition of the QM region (not only its size), and a convergence test is proposed to be a part of setting up QM/MM simulations.

Bending of DNA duplexes with mutation motifs.

Mutations can be induced by environmental factors but also arise spontaneously during DNA replication or due to deamination of methylated cytosines at CpG dinucleotides. Sites where mutations occur with higher frequency than would be expected by chance are termed hotspots while sites that contain mutations rarely are termed coldspots. Mutations are permanently scanned and repaired by repair systems. Among them, the mismatch repair targets base pair mismatches, which are discriminated from canonical base pairs by probing altered elasticity of DNA. Using biased molecular dynamics simulations, we investigated the elasticity of coldspots and hotspots motifs detected in human genes associated with inherited disorders, and also of motifs with Czech population hotspots and de novo mutations. Main attention was paid to mutations leading to G/T and A+/C pairs. We observed that hotspots without CpG/CpHpG sequences are less flexible than coldspots, which indicates that flexible sequences are more effectively repaired. In contrary, hotspots with CpG/CpHpG sequences exhibited increased flexibility as coldspots. Their mutability is more likely related to spontaneous deamination of methylated cytosines leading to C > T mutations, which are primarily targeted by base excision repair. We corroborated conclusions based on computer simulations by measuring melting curves of hotspots and coldspots containing G/T mismatch.

Molecular dynamics study of Pseudomonas aeruginosa lectin-II complexed with monosaccharides.

We present the results of a series of 10-ns molecular dynamics simulations on Pseudomonas aeruginosa lectin-II (PA-IIL) and its complexes with four different monosaccharides. We compare the saccharide-free, saccharide-occupied, and saccharide- and ion-free forms of the lectin. The results are coupled with analysis of the water density map and calcium coordination. The water density pattern around the binding site in the free lectin molecular dynamics was fitted with that in the X-ray and with the hydroxyl groups of the monosaccharide within the lectin/monosaccharide complexes and the best ligand was predicted based on the best fit. Interestingly, the water density pattern around the binding site in the uncomplexed lectin exactly fitted the O2, O3, and O4 hydroxyl groups of the fucose complex with the lectin. This observation could lead to a hypothesis that the replacement of these three water molecules from the binding site by the monosaccharide decreases the entropy of the complex and increases the entropy of the water molecules, which favors the binding. It suggests that the high density peaks of the solvent around the binding site in the free protein could be the tool to predict hydroxyl group orientation of the sugar in the protein/sugar complexes. The high affinity of PA-IIL binding site is also attributed to the presence of two calcium ions, each of them making five to six coordinations with the protein part and two coordinations with either water or the monosaccharide. When the calcium ions are removed from the simulated system, they are replaced by sodium ions from the solvent. These observations rationalize the high binding affinity of PA-IIL towards fucose.

Mechanism of hydrogen-bond array isomerization in tetrahydroxycalix[4]arene and tetrahydroxythiacalix[4]arene.

Possible rearrangement mechanisms of hydrogen-bond arrays formed at the lower rim of tetrahydroxycalix[4]arene and tetrahydroxythiacalix[4]arene were studied by means of density functional theory and the resolution identity approximation modification of Møller-Plesset perturbation theory (RI-MP2). Influence of solvent to height of energy barriers was quantified by use of the conductorlike screening model (COSMO) of implicit solvent (chloroform). Generally, two types of mechanisms were investigated. The first is represented by a synchronous single-step jump of all four hydroxyl protons. Pathways of the second mechanism include the rotation of one or more hydroxyl groups around the CAr-O bond. Theoretical results, in agreement with recently published experimental data (Lang et al. J. Chem. Phys. 2005, 122, 044056), prefer a jump mechanism for the methylene-bridged calix[4]arene. Concerning the thiacalix[4]arene, results obtained by COSMO as well as RI-MP2 calculations show that the rotational mechanism is very competitive and it could even be more favorable.

Conformations of flanking bases in HIV-1 RNA DIS kissing complexes studied by molecular dynamics.

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.

Behavior of BsoBI endonuclease in the presence and absence of DNA.

BsoBI is a type II restriction endonuclease belonging to the EcoRI family. There is only one previously published X-ray structure for this endonuclease: it shows a homodimer of BsoBI completely encircling DNA in a tunnel. In this work, molecular dynamics simulations were employed to elucidate possible ways in which DNA is loaded into this complex prior to its cleavage. We found that the dimer does not open spontaneously when DNA is removed from the complex on the timescale of our simulations (~ 0.5 µs). A biased simulation had to be used to facilitate the opening, which revealed a possible way for the two catalytic domains to separate. The α-helices connecting the catalytic and helical domains were found to act as a hinge during the separation. In addition, we found that the opening of the BsoBI dimer was influenced by the type of counterions present in the environment. A reference simulation of the BsoBI/DNA complex further showed spontaneous reorganization of the active sites due to the binding of solvent ions, which led to an active-site structure consistent with other experimental structures of type II restriction endonucleases determined in the presence of metal ions.