Strong Patterns in Homooligomer Tracts Occurrences in Non-Coding and in Potential Regulatory Sites in Eukaryotic Genomes

Abstract

Previous studies of the dinucleotides flanking both the 5′ and 3′ ends of homooligomer tracts have shown that some flanks are consistently preferred over others (1,2). In the first preferred group, the homooligomer tracts are flanked by the same nucleotide and/or the complementary nucleotides, e.g., ATA_n, TTA_n, CCG_n, where n=2–5. Runs flanked by nucleotides with which they cannot base pair are distinctly disfavored. (In this group A/T_n are flanked by C and/or G; G_n/C_n are flanked by A/T, e.g., CGA_n, T_nGG, G., AT). The frequencies of runs flanked by AorT, and G or C (“mixed” group) are as expected. Here we seek the origin of this effect and its relevance to protein-DNA interactions. Surprisingly, within the first group, runs flanked by their complements with a pyrimidine-purine junction (e.g., TTA_n, C_nGG) are greatly preferred. The frequencies of their purine-pyrimidine junction mirror-images is just as expected. This effect, as well as additional ones enumerated below, is seen universally in eukaryotes and in prokaryotes, although it is stronger in the former. Detailed analysis of regulatory regions shows these strong trends, particularly in GC sequences. The potential relationship to DNA conformation and DNA-protein interaction is discussed.     

164 Extracting dynamics information from multiple structures

Meaningful dynamics information can be extracted from multiple experimental structures of the same, or closely related, proteins or RNAs. The covariance matrix of atom positions is decomposable into its principal components, and in this way, it is possible to rank-order the changes in the set of structures, and to determine what the most significant changes are. Usually, only a few principal components dominate the motions of the structures, and these usually relate to the functional dynamics. This dynamics information provides strong evidence for the plasticity of protein and RNA structures, and also suggests that these structures almost always have a highly limited repertoire of motions. In some cases, such as HIV protease, the dominant motions are opening and closing over the active site. For myoglobin, the changes are much smaller, reflecting in part the small changes in sequence, but nonetheless they show characteristic details that depend on the species. Sets of structures can also be used to derive the effective microscopic forces that are forcing a given conformational transition.     

Relationship Between Curved DNA Conformations and Slow Gel Migration

Abstract

We propose some specific DNA conformations that explain, in terms of molecular conformations, the anomalous gel electrophoretic behavior of the sequences (VA₄T₄X)₁, and (V₂A₃X₂)₁ where V and X are either G or C. Previously (J. Biomole. Struct. Dyn. 4, 41, 1986) we considered hydrophobic interactions a mong aliphatic hydrocarbon groups in A/T sequences. In the sequences (T)_n · (A)_n, the T's are slightly bent to yield structures with tightly stacked methyl groups along one side of the major groove. By folding together the two pairs of stacked methyls on the opposite sides of the major groove, TTAA might yield a relatively sharp bend. On this basis, we show below that the sequences (VT₄A₄X)₁ might form a very tightly coiled super-helix whereas the sequences (VA₄T₄X)₁ form a broad super-helix of radius ~ 120 A for i = 25. The sequence (V₂A₃T₃X₂)₁ forms a slightly smaller radius super-helix. The time of passage through the gel has been taken to be inversely proportional to the smallesuiimension of the molecule. Specifically we are taking the ratio of the apparent molecular weight to the actual molecular weight to be related to the moment of inertia I₁ about the smallest principal axis of the molecular conformation. We find a good fit to the experimental gel mobility data of Hagerman (2) if we assume this ratio to be proportional to (I₁)^1/5.     

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases

Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.     

Coupling dynamics and evolutionary information with structure to identify protein regulatory and functional binding sites

Binding sites in proteins can be either specifically functional binding sites (active sites) that bind specific substrates with high affinity or regulatory binding sites (allosteric sites), that modulate the activity of functional binding sites through effector molecules. Owing to their significance in determining protein function, the identification of protein functional and regulatory binding sites is widely acknowledged as an important biological problem. In this work, we present a novel binding site prediction method, Active and Regulatory site Prediction (AR-Pred), which supplements protein geometry, evolutionary, and physicochemical features with information about protein dynamics to predict putative active and allosteric site residues. As the intrinsic dynamics of globular proteins plays an essential role in controlling binding events, we find it to be an important feature for the identification of protein binding sites. We train and validate our predictive models on multiple balanced training and validation sets with random forest machine learning and obtain an ensemble of discrete models for each prediction type. Our models for active site prediction yield a median area under the curve (AUC) of 91% and Matthews correlation coefficient (MCC) of 0.68, whereas the less well-defined allosteric sites are predicted at a lower level with a median AUC of 80% and MCC of 0.48. When tested on an independent set of proteins, our models for active site prediction show comparable performance to two existing methods and gains compared to two others, while the allosteric site models show gains when tested against three existing prediction methods. AR-Pred is available as a free downloadable package at https://github.com/sambitmishra0628/AR-PRED_source .     

Myosin flexibility: structural domains and collective vibrations

The movement of the myosin motor along an actin filament involves a directed conformational change within the cross-bridge formed between the protein and the filament. Despite the structural data that has been obtained on this system, little is known of the mechanics of this conformational change. We have used existing crystallographic structures of three conformations of the myosin head, containing the motor domain and the lever arm, for structural comparisons and mechanical studies with a coarse-grained elastic network model. The results enable us to define structurally conserved domains within the protein and to better understand myosin flexibility. Notably they point to the role of the light chains in rigidifying the lever arm and to changes in flexibility as a consequence of nucleotide binding.     

Predicting binding sites of hydrolase-inhibitor complexes by combining several methods

Background

Protein-protein interactions play a critical role in protein function. Completion of many genomes is being followed rapidly by major efforts to identify interacting protein pairs experimentally in order to decipher the networks of interacting, coordinated-in-action proteins. Identification of protein-protein interaction sites and detection of specific amino acids that contribute to the specificity and the strength of protein interactions is an important problem with broad applications ranging from rational drug design to the analysis of metabolic and signal transduction networks.

Results

In order to increase the power of predictive methods for protein-protein interaction sites, we have developed a consensus methodology for combining four different methods. These approaches include: data mining using Support Vector Machines, threading through protein structures, prediction of conserved residues on the protein surface by analysis of phylogenetic trees, and the Conservatism of Conservatism method of Mirny and Shakhnovich. Results obtained on a dataset of hydrolase-inhibitor complexes demonstrate that the combination of all four methods yield improved predictions over the individual methods.

Conclusions

We developed a consensus method for predicting protein-protein interface residues by combining sequence and structure-based methods. The success of our consensus approach suggests that similar methodologies can be developed to improve prediction accuracies for other bioinformatic problems.     

Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia

Vasodilatory responses to exogenous nitric oxide (NO) are diminished following exposure to chronic hypoxia (CH) in isolated, perfused rat lungs. We hypothesized that both endothelium-derived reactive oxygen species (ROS) and endothelin-1 (ET-1) mediate this attenuated NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined vasodilatory and vascular smooth muscle (VSM) Ca2+ responses to the NO donor spermine NONOate in UTP-constricted, isolated pressurized small pulmonary arteries from control and CH rats. Consistent with our previous findings in perfused lungs, we observed attenuated NO-dependent vasodilation following CH in endothelium-intact vessels. However, in endothelium-denuded vessels, responses to spermine NONOate were augmented in CH rats compared with controls, thus demonstrating an inhibitory influence of the endothelium on NO-dependent reactivity following CH. Whereas both the ROS scavenger tiron and the ETA receptor antagonist BQ-123 augmented NO-dependent reactivity in endothelium-intact vessels from CH rats, neither fully restored vasodilatory responses to those observed following endothelium denudation in vessels from CH rats. In contrast, the combination of tiron and BQ-123 or the nonselective ET receptor antagonist PD-145065 enhanced NO responsiveness in endothelium-intact vessels from CH rats similar to that observed following endothelium denudation. We conclude that both endothelium-derived ROS and ET-1 attenuate NO-dependent pulmonary vasodilation following CH. Furthermore, CH augments pulmonary VSM reactivity to NO.     

Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia

Chronic hypoxia (CH) increases pulmonary arterial endothelial nitric oxide (NO) synthase (NOS) expression and augments endothelium-derived nitric oxide (EDNO)-dependent vasodilation, whereas vasodilatory responses to exogenous NO are attenuated in CH rat lungs. We hypothesized that reactive oxygen species (ROS) inhibit NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined responses to the EDNO-dependent vasodilator endothelin-1 (ET-1) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) in isolated lungs from control and CH rats in the presence or absence of ROS scavengers under normoxic or hypoxic ventilation. NOS was inhibited in lungs used for SNAP experiments to eliminate influences of endogenously produced NO. Additionally, dichlorofluorescein (DCF) fluorescence was measured as an index of ROS levels in isolated pressurized small pulmonary arteries from each group. We found that acute hypoxia increased DCF fluorescence and attenuated vasodilatory responses to ET-1 in lungs from control rats. The addition of ROS scavengers augmented ET-1-induced vasodilation in lungs from both groups during hypoxic ventilation. In contrast, upon NOS inhibition, DCF fluorescence was elevated and SNAP-induced vasodilation diminished in arteries from CH rats during normoxia, whereas acute hypoxia decreased DCF fluorescence, which correlated with augmented reactivity to SNAP in both groups. ROS scavengers enhanced SNAP-induced vasodilation in normoxia-ventilated lungs from CH rats similar to effects of hypoxic ventilation. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation.