Effect of a U:G mispair on the water around DNA.

DNA repair proteins are able to discriminate DNA lesions among an abundance of intact DNA with high selectivity. To investigate detectable characteristics of one specific lesion, we compare statistical results from molecular dynamics simulations of two different DNA in water, one with an intact C:G pair and one that contains a U:G mispair, and perform a comparative analysis of the water dynamics around the two. Our data show that in addition to the local DNA conformation, also the surrounding water shell exhibits significant differences that may help mispair discrimination. The chemical groups which account for a U:G mispair to exhibit a wobble conformation instead of the 'proper' Watson-Crick pairing of a C:G pair, that is an oxygen atom (in uracil) instead of an amino group (in cytosine), also order the water molecules around the bases such that they act predominantly as hydrogen-bond donor or acceptor to the uracil or cytosine base, respectively. These changes in water conformation stretch into the second solvation shell, which may be exploited by repair enzymes to achieve lesion detection with high efficiency.

The relation between lignin sequence and its 3D structure.

BACKGROUND: Lignin, the second most abundant biopolymer on earth, plays a major structural role in plants, conferring mechanical strength and regulating water conduction. Understanding the three-dimensional structure of lignin is important for fundamental reasons as well as engineering plants towards lignin valorization. Lignin lacks a specific primary sequence, making its average chemical composition the focus of most recent studies. However, it remains unclear whether the 3D structure of lignin molecules depends on their sequence. METHODS: We performed all-atom molecular dynamics simulation of three S/G-lignin molecules with the same average composition but different sequence. RESULTS: A detailed statistical analysis of the radius of gyration and relative shape anisotropy reveals that the lignin sequence has no statistically significant effect on the global three-dimensional structure. We found however, that homopolymers of C-lignin with the same molecular weight have smaller radii of gyration than S/G-lignin. We attribute this to lower hydroxyl content of C-lignin, which makes it more compact and rigid. CONCLUSIONS: The 3D structure of lignin is influenced by the overall content of monomeric units and interunit linkages and not by its precise primary sequence. GENERAL SIGNIFICANCE: Lignin is assumed to not have a well-defined primary structure. The results presented here demonstrate there are no significant differences in the global 3D structure of lignin molecules with the same average composition but different primary sequence.

Novel multi-target compounds in the quest for new chemotherapies against Alzheimer's disease: An experimental and theoretical study.

The lack of any effective therapy along with the aging world population anticipates a growth of the worldwide incidence of Alzheimer's disease (AD) to more than 100 million cases by 2050. Accumulation of extracellular amyloid-ß (Aß) plaques, intracellular tangles in the brain, and formation of reactive oxygen species (ROS) are the major hallmarks of the disease. In the amyloidogenic process, a ß-secretase, known as BACE 1, plays a fundamental role in the production of Aß fragments, and therefore, inhibition of such enzymes represents a major strategy for the rational design of anti-AD drugs. In this work, a series of four multi-target compounds (1-4), inspired by previously described ionophoric polyphenols, have been synthesized and studied. These compounds have been designed to target important aspects of AD, including BACE 1 enzymatic activity, Aß aggregation, toxic concentrations of Cu2+ metal ions and/or ROS production. Two other compounds (5 and 6), previously reported by some of us as antimalarial agents, have also been studied because of their potential as multi-target species against AD. Interestingly, compounds 3 and 5 showed moderate to good ability to inhibit BACE 1 enzymatic activity in a FRET assay, with IC50's in the low micromolar range (4.4 ±â€¯0.3 and 1.7 ±â€¯0.3 µM, respectively), comparable to other multi-target species, and showing that the observed activity was in part due to a competitive binding of the compounds at the active site of the enzyme. Theoretical docking calculations overall agreed with FRET assay results, displaying the strongest binding affinities for 3 and 5 at the active site of the enzyme. In addition, all compounds selectively interacted with Cu2+ metal ions forming 2:1 complexes, inhibited the production of Aß-Cu2+ catalyzed hydroxyl radicals up to a ∼100% extent, and scavenged AAPH-induced peroxyl radical species comparably to resveratrol, a compound used as reference in this work. Our results also show good anti-amyloidogenic ability: compounds 1-6 inhibited both the Cu2+-induced and self-induced Aß(1-40) fibril aggregation to an extent that ranged from 31% to 77%, while they disaggregated pre-formed Aß(1-40) mature fibrils up to a 37% and a 69% extent in absence and presence of Cu2+, respectively. Cytotoxicity was additionally studied in Tetrahymena thermophila and HEK293 cells, and compared to that of resveratrol, showing that compounds 1-6 display lower toxicity than that of resveratrol, a well-known non-toxic polyphenol.

RAG-3D: a search tool for RNA 3D substructures.

To address many challenges in RNA structure/function prediction, the characterization of RNA's modular architectural units is required. Using the RNA-As-Graphs (RAG) database, we have previously explored the existence of secondary structure (2D) submotifs within larger RNA structures. Here we present RAG-3D-a dataset of RNA tertiary (3D) structures and substructures plus a web-based search tool-designed to exploit graph representations of RNAs for the goal of searching for similar 3D structural fragments. The objects in RAG-3D consist of 3D structures translated into 3D graphs, cataloged based on the connectivity between their secondary structure elements. Each graph is additionally described in terms of its subgraph building blocks. The RAG-3D search tool then compares a query RNA 3D structure to those in the database to obtain structurally similar structures and substructures. This comparison reveals conserved 3D RNA features and thus may suggest functional connections. Though RNA search programs based on similarity in sequence, 2D, and/or 3D structural elements are available, our graph-based search tool may be advantageous for illuminating similarities that are not obvious; using motifs rather than sequence space also reduces search times considerably. Ultimately, such substructuring could be useful for RNA 3D structure prediction, structure/function inference and inverse folding.

Computational prediction of riboswitch tertiary structures including pseudoknots by RAGTOP: a hierarchical graph sampling approach.

The modular organization of RNA structure has been exploited in various computational and theoretical approaches to identify RNA tertiary (3D) motifs and assemble RNA structures. Riboswitches exemplify this modularity in terms of both structural and functional adaptability of RNA components. Here, we extend our computational approach based on tree graph sampling to the prediction of riboswitch topologies by defining additional edges to mimick pseudoknots. Starting from a secondary (2D) structure, we construct an initial graph deduced from predicted junction topologies by our data-mining algorithm RNAJAG trained on known RNAs; we sample these graphs in 3D space guided by knowledge-based statistical potentials derived from bending and torsion measures of internal loops as well as radii of gyration for known RNAs. We present graph sampling results for 10 representative riboswitches, 6 of them with pseudoknots, and compare our predictions to solved structures based on global and local RMSD measures. Our results indicate that the helical arrangements in riboswitches can be approximated using our combination of modified 3D tree graph representations for pseudoknots, junction prediction, graph moves, and scoring functions. Future challenges in the field of riboswitch prediction and design are also discussed.

Increasing cleavage specificity and activity of restriction endonuclease KpnI.

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg(2+) ion higher than 500 µM mediates promiscuous activity, Ca(2+) suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca(2+)-mediated cleavage but imparting high cleavage fidelity with Mg(2+). High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca(2+)-mediated cleavage activity by altering the geometry of the Ca(2+)-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.

Predicting helical topologies in RNA junctions as tree graphs.

RNA molecules are important cellular components involved in many fundamental biological processes. Understanding the mechanisms behind their functions requires knowledge of their tertiary structures. Though computational RNA folding approaches exist, they often require manual manipulation and expert intuition; predicting global long-range tertiary contacts remains challenging. Here we develop a computational approach and associated program module (RNAJAG) to predict helical arrangements/topologies in RNA junctions. Our method has two components: junction topology prediction and graph modeling. First, junction topologies are determined by a data mining approach from a given secondary structure of the target RNAs; second, the predicted topology is used to construct a tree graph consistent with geometric preferences analyzed from solved RNAs. The predicted graphs, which model the helical arrangements of RNA junctions for a large set of 200 junctions using a cross validation procedure, yield fairly good representations compared to the helical configurations in native RNAs, and can be further used to develop all-atom models as we show for two examples. Because junctions are among the most complex structural elements in RNA, this work advances folding structure prediction methods of large RNAs. The RNAJAG module is available to academic users upon request.

The effect of a G:T mispair on the dynamics of DNA.

Distortions in the DNA sequence such as damages or mispairs are specifically recognized and processed by DNA repair enzymes. A particular challenge for the enzymatic specificity is the recognition of a wrongly-placed native nucleotide such as thymine in T:G mispairs. An important step of substrate binding which is observed in many repair proteins is the flipping of the target base out of the DNA helix into the enzyme's active site. In this work we investigate how much the intrinsic dynamics of mispaired DNA is changed compared to canonical DNA. Our molecular dynamics simulations of DNA with and without T:G mispairs show significant differences in the conformation of paired and mispaired DNA. The wobble pair T:G shows local distortions such as twist, shear and stretch which deviate from canonical B form values. Moreover, the T:G mispair is found to be kinetically less stable, exhibiting two states with respect to base opening: a closed state comparable to the canonical base pairs, and a more open state, indicating a proneness for base flip. In addition, we observe that the thymine base in a T:G mispair is significantly more probable to be flipped than thymine in a T:A pair or cytosine in a C:G pair. Such local deformations and in particular the existence of a second, more-open state can be speculated to help the target-site recognition by repair enzymes.

Role of magnesium ions in DNA recognition by the EcoRV restriction endonuclease.

The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, Mg(A)(2+), binds to the phosphate group where the cleavage occurs and is required for catalysis, but the role of the other ion, Mg(B)(2+) is debated. Here, multiple independent molecular dynamics simulations suggest that Mg(B)(2+) is crucial for achieving a tightly bound protein-DNA complex and stabilizing a conformation that allows cleavage. In the absence of Mg(B)(2+) in all simulations the protein-DNA hydrogen bond network is significantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely.

Magnesium-dependent active-site conformational selection in the Diels-Alderase ribozyme.

The Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme, accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition, a reaction with broad application in organic chemistry. Here, the Diels-Alderase ribozyme is examined via molecular dynamics (MD) simulations in both crystalline and aqueous solution environments. The simulations indicate that the catalytic pocket is highly dynamic. At low Mg(2+) ion concentrations, inactive states with the catalytic pocket closed dominate. Stabilization of the enzymatically active, open state of the catalytic pocket requires a high concentration of Mg(2+) ions (e.g., 54 mM), with cations binding to specific phosphate sites on the backbone of the residues bridging the opposite strands of the pocket. The free energy profile for pocket opening at high Mg(2+) cation concentration exhibits a double minimum, with a barrier to opening of approximately 5.5 kJ/mol and the closed state approximately 3 kJ/mol lower than the open state. Selection of the open state on substrate binding leads to the catalytic activity of the ribozyme. The simulation results explain structurally the experimental observation that full catalytic activity depends on the Mg(2+) ion concentration.

Mechanism of DNA recognition by the restriction enzyme EcoRV.

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV-cognate DNA crystallographic complex, a sharp kink of 50 degrees has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GA(CH(3))TATC (TA-CH(3)). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV-DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage.

Structural and electrostatic properties of ubiquitination and related pathways.

Post-translational modification by ubiquitin and ubiquitin-like (UBL) proteins is a key mechanism for cellular control. The specificity of the enzymes of ubiquitination and their close paralogs is dependent on their molecular electrostatic potentials. For example, analysis of molecular electrostatic potentials and electrostatically key residues can account for the selectivity of different E1s (activating enzymes) and of different SUMO proteases. The molecular interactions of the ubiquitin conjugating enzymes, the ubiquitin family proteins (UFP) and UBL domains are discussed in detail. An interesting observation is that the Non Canonical Ubiquitin Conjugating Enzymes (NCUBEs) have electrostatic potentials that are more similar to the UBC9 orthologs, the SUMO conjugating enzymes, than they are to other ubiquitin conjugating enzymes. It had previously been suggested that UBC9 may select for SUMO based on its difference in electrostatic potential as compared to other E2s but the NCUBE exception suggests that this may not be the case. The web site http://www.ubiquitin-resource.org/ allows users to find the E2s most electrostatically similar to a query E2. Where possible, models have been made for all E2 domains in the SMART database (http://smart.embl-heidelberg.de/). A brief overview of molecular electrostatic potentials and their application to understanding protein function is also given.