Article
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine ring of the substrate, dUMP, and the pterine ring of the cofactor analogue, THF, in the active site of the phosphorylated enzyme. The ED analysis indicates changes in the behavior of collective motions in the phosphorylated enzyme, suggesting that the formation of the closed ternary complex is hindered. Computed free energies, in agreement with structural analysis, predict that the binding of dUMP and THF to hTS is favored in the native compared to phosphorylated state of the enzyme. The paper describes at the structural level how phosphorylation at the distant site influences the ligand binding. We propose that the 'phosphorylation effect' is transmitted from the outside loop of Ser 124 into the active site via a subtle mechanism initiated by the long-range electrostatic repulsion between the phosphate groups of dUMP and Ser124. The mechanism can be described in terms of the interplay between the two groups of amino acids: the link (residues 125-134) and the patch (residues 189-192), resulting in the change of orientation of the pyrimidine ring of dUMP, which, in turn, prevents the correct alignment between the latter ring and the pterin ring of THF.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... The simulations were started from the monomer A of the dimeric crystallographic structure of the ternary complex of hTS with dUMP and Tomudex (PDB code: 1I00) [16], from which the residue numbering used throughout the paper has been adopted. The monomer A of the 1I00 structure has been prepared for the simulations according to the protocol described in Jarmuła et al. [17], with the Tomudex molecule being replaced with the molecule of tetrahydrofolate (THF; close analogue of meTHF), resulting in the hTS-dUMP-THF ternary system. Each time one of the Tyr 33, 65, 135, 213, 230, 258, and 301 residues was substituted at the ortho carbon of the phenolic ring with the-NO 2 group, leading to nitrated variants of the hTS-dUMP-THF system. ...
... The protocol for energy minimization and molecular dynamics runs was the same as the one used in Jarmuła et al. [17]. Briefly, it consisted of (1) the steepest descent, followed by conjugate gradients energy minimization, (2) gradual heating to 300 K, (3) equilibration for 199 ps, and (4) data collection runs (DCR) for 2,000 ps. ...
... Relative solvent accessibility shown on the LIGPLOT plots was evaluated with the NACCESS program [28]. Average structures from the MD trajectories were calculated using the frames taken from the last 1,200 ps of DCR and applying the protocol used in Jarmuła et al. [17]. ...
Article
Full-text available
Tyrosine nitration is a widespread post-translational modification capable of affecting both the function and structure of the host protein molecule. Enzyme thymidylate synthase (TS), a homodimer, is a molecular target for anticancer therapy. Recently purified TS preparations, isolated from mammalian tissues, were found to be nitrated, suggesting this modification to appear endogenously in normal and tumor tissues. Moreover, human TS (hTS) nitration in vitro led to a by twofold lowered catalytic activity following nitration in average of 1 tyrosine residue per monomer (Dąbrowska-Maś et al. in Org Biomol Chem 10:323-331, 2012), with the modification identified by mass spectrometry at seven different sites (Y33, Y65, Y135, Y213, Y230, Y258 and Y301). In the present paper, combined computational approach, including molecular and essential dynamics and free energy computations, was used to predict the influence on the activity of hTS of nitration of each of the seven tyrosine residues. The simulations were based on the crystal structure of hTS ternary complex with dUMP and Tomudex (PDB code: 1I00), with the Tomudex molecule replaced by the molecule of TS cofactor analogue, tetrahydrofolate. The present results indicate that while with nitration of five out of seven residues (Y33, Y135, Y230, Y258 and Y301), single residue modification appears to have a strong reducing effect on the activity, with the remaining two, Y65 and Y213, no or a weaker influence is apparent. Taken together, these results demonstrate that tyrosine nitrations in the hTS enzyme show clear tendency to influence the structure and dynamics and, in turn, catalytic properties of the host enzyme. These effects are overall distance-dependent.
... These structural rearrangements allow protein multimerization (small HSPs [4][5][6]), protein ordering (Microtubule Associated Protein Tau [7]), and protein-protein interactions (p47 [8]). Moreover, phosphorylation can affect the enzyme activity endorsing the ligand binding in the active site (Thymidylate synthase) [9,10]. Frequently, the modification of multiple phosphorylation sites of a protein constitutes more than an on/off mechanism, since the level of phosphorylation can induce threshold related events. ...
... This representation returns the probability of finding the system in a particular state characterized by a combination of two reaction-coordinates values. The first two principal components of motion are the considered reaction-coordinates. From this analysis, the minimum frequency value (8), or better the probability of the most likely conformation, and the number of minima over a case dependent threshold of frequency (9) are collected. The latter is obtained applying a 20x20 points grid and summing the values included in a 3x3 box: boxes with values over the threshold are considered. ...
Article
Full-text available
Phosphorylation is one of the most important post-translational modifications (PTM) employed by cells to regulate several cellular processes. Studying the effects of phosphorylations on protein structures allows to investigate the modulation mechanisms of several proteins including chaperones, like the small HSPs, which display different multimeric structures according to the phosphorylation of a few serine residues. In this context, the proposed study is aimed at finding a method to correlate different PTM patterns (in particular phosphorylations at the monomers interface of multimeric complexes) with the dynamic behaviour of the complex, using physicochemical parameters derived from molecular dynamics simulations in the timescale of nanoseconds. We have developed a methodology relying on computing nine physicochemical parameters, derived from the analysis of short MD simulations, and combined with N identifiers that characterize the PTMs of the analysed protein. The nine general parameters were validated on three proteins, with known post-translational modified conformation and unmodified conformation. Then, we applied this approach to the case study of αB-Crystallin, a chaperone which multimeric state (up to 40 units) is supposed to be controlled by phosphorylation of Ser45 and Ser59. Phosphorylation of serines at the dimer interface induces the release of hexamers, the active state of αB-Crystallin. 30 ns of MD simulation were obtained for each possible combination of dimer phosphorylation state and average values of structural, dynamic, energetic and functional features were calculated on the equilibrated portion of the trajectories. Principal Component Analysis was applied to the parameters and the first five Principal Components, which summed up to 84 % of the total variance, were finally considered. The validation of this approach on multimeric proteins, which structures were known both modified and unmodified, allowed us to propose a new approach that can be used to predict the impact of PTM patterns in multi-modified proteins using data collected from short molecular dynamics simulations. Analysis on the αB-Crystallin case study clusters together all-P dimers with all-P hexamers and no-P dimer with no-P hexamer and results suggest a great influence of Ser59 phosphorylation on chain B.
... The reason for this discrepancy is not known but other factors such as post-translation modifications may affect TS activity. As shown in earlier studies, phosphorylation on Ser-12 [30] or S-glutathionylation on Cys-195 lead to decreased activity [31]. ...
... D). The lack of correlation between the levels of mRNA and protein may be explained by post-translational modifications such as S-glutathionylation affecting protein stability [30,31,39]. ...
Article
Full-text available
Background: Deficiency in thymidine kinase 2 (TK2) or p53 inducible ribonucleotide reductase small subunit (p53R2) is associated with tissue specific mitochondrial DNA (mtDNA) depletion. To understand the mechanisms of the tissue specific mtDNA depletion we systematically studied key enzymes in dTMP synthesis in mitochondrial and cytosolic extracts prepared from adult rat tissues. Results: In addition to mitochondrial TK2 a cytosolic isoform of TK2 was characterized, which showed similar substrate specificity to the mitochondrial TK2. Total TK activity was highest in spleen and lowest in skeletal muscle. Thymidylate synthase (TS) was detected in cytosols and its activity was high in spleen but low in other tissues. TS protein levels were high in heart, brain and skeletal muscle, which deviated from TS activity levels. The p53R2 proteins were at similar levels in all tissues except liver where it was ~ 6-fold lower. Our results strongly indicate that mitochondria in most tissues are capable of producing enough dTTP for mtDNA replication via mitochondrial TK2, but skeletal muscle mitochondria do not and are most likely dependent on both the salvage and de novo synthesis pathways. Conclusion: These results provide important information concerning mechanisms for the tissue dependent variation of dTTP synthesis and explained why deficiency in TK2 or p53R2 leads to skeletal muscle dysfunctions. Furthermore, the presence of a putative cytosolic TK2-like enzyme may provide basic knowledge for the understanding of deoxynucleoside-based therapy for mitochondrial disorders.
... To assess the potential of phosphorylation of different histidine residues on the catalytic activity of TS, molecular dynamics (MD) studies were carried out (for MD simulation protocol see [31,32]). Candidates for phosphorylation were selected, based on initial results (some of them not finally confirmed) of MS analysis of different protein samples. Both the N1-and N3-phosphorylated variants of certain histidine residues were selected for simulations, since they are indistinguishable by MS, and NMR analysis showed that both variants were present in phosphorylated samples. ...
... The enriched phosphorylated fractions of recombinant TS proteins, albeit representing in each case only ≈1% of the total purified TS protein, provided enough protein (with each TS about 1 mg) to attempt 31 P NMR qualitative identification of phosphorylated amino acid residue(s). An unexpected finding of phosphorus in a phosphoramidate (acid-labile) bond, pointing to the modification of histidine residue(s), raises the question as to whether a similar modification may be present in the endogenous mammalian TS proteins studied. ...
... The phosphorylation lowered TS catalytic activity and affected binding of the TS inhibitor, 5-fluorodeoxyuridylate (FdUMP). The results of molecular dynamics simulations have shown that CK2-mediated phosphorylation of serine 124 residue of the human TS leads to a protein conformational change, resulting in an unfavorable position of the substrate (dUMP) and cofactor (methylene-THF) in the active center (Jarmuła et al., 2010). In addition, a stiffening of certain protein fragments, especially the loop closing the active center pocket, has been shown . ...
Article
Full-text available
Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.
... In view of the difficulties in the analysis of phosphorylation of basic amino acids, the usage of in silico simulations should be considered useful, for example to predict the influence of phosphorylation on protein properties. Such a study with the use of molecular dynamics simulations allowed revealing the mechanism of the influence of Ser124 phosphorylation on the catalytic activity of human thymidylate synthase (Jarmuła et al., 2010), and the same approach could be applied to study the consequences of phosphorylation of basic amino acid residues, taking advantage of the force-field parameters calculated recently for phosphohistidine (Kosinsky et al., 2004; Homeyer et al., 2006). ...
Article
Full-text available
Reversible phosphorylation is the most widespread posttranslational protein modification, playing regulatory role in almost every aspect of cell life. The majority of protein phosphorylation research has been focused on serine, threonine and tyrosine that form acid-stable phosphomonoesters. However, protein histidine, arginine and lysine residues also may undergo phosphorylation to yield acid-labile phosphoramidates, most often remaining undetected in conventional studies of protein phosphorylation. It has become increasingly evident that acid-labile protein phosphorylations play important roles in signal transduction and other regulatory processes. Beside acting as high-energy intermediates in the transfer of the phosphoryl group from donor to acceptor molecules, phosphohistidines have been found so far in histone H4, heterotrimeric G proteins, ion channel KCa3.1, annexin 1, P-selectin and myelin basic protein, as well as in recombinant thymidylate synthase expressed in bacterial cells. Phosphoarginines occur in histone H3, myelin basic protein and capsidic protein VP12 of granulosis virus, whereas phospholysine in histone H1. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated.
... In several cases, enzyme activity or protein function has been reported to be regulated by inhibitory or activatory phosphorylation events at specific protein sites. At the molecular level, these events are mediated by electrostatic repulsion between the phospho-residues and the neighboring negatively charged aminoacids [46,47,48,49,50,51], pointing out a general and important regulatory mechanism. These mechanisms can be successfully investigated in atomic details by MD simulations [47,52]. ...
Article
Full-text available
E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.
... On phosphorylation, the N-terminal region containing the phosphorylated serine residue changed conformation and moved approximately 35Å35˚35Å, which was accompanied by changes in the tertiary and quaternary structures, leading to activation [60]. In the case of human thymidylate synthase, phosphorylation of a serine residue that is situated far away from the catalytic site (23Å23˚23Å) inhibited the enzyme by changing orientation of the substrate, probably through a long-range electrostatic repulsion [61]. Phosphorylation of the activation loop in protein kinases can be regarded as another example where phosphorylation of a far site affected the enzymatic activity [53]. ...
Article
We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram-negative eubacterium Thermus thermophilus HB8 using gel-free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 Å of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilus HB8.
... Cette phosphorylation diminuerait la capacité de l'enzyme à se lier à ses deux ligands, le dUMP et le THF, par répulsion électrostatique entre le phosphate du dUMP et celui de la Ser 124 (Jarmuła et al., 2010). La phosphorylation de la Ser 124 pourrait ainsi être responsable de l'asymétrie des sous-unités de la TS dans leur capacité à lier les substrats (Figure 22). ...
Thesis
Full-text available
La O-GlcNAcylation (O-N-acétylglucosaminylation) est une MPT (modification post-traductionnelle) dynamique et réversible catalysée par un unique couple d’enzymes antagonistes : l’OGT (O-GlcNAc transférase) et l’OGA (O GlcNAcase). Elle est considérée comme un véritable senseur nutritionnel et régule un grand nombre de mécanismes cellulaires fondamentaux. En ciblant des oncoprotéines et des suppresseurs de tumeur, sa dérégulation est associée à la cancérogenèse et la progression tumorale. En revanche, son rôle dans la réponse aux thérapies anti-cancéreuses est très peu étudié. Il a été néanmoins montré récemment que l’hyper-O-GlcNAcylation impacte la réponse de certains cancers à des drogues telles que le tamoxifène, le cisplatine, le bortézomib et le 5-FU (5-fluorouracile). Le 5-FU est la chimiothérapie de référence du CCR (cancer colorectal) et la TS (Thymidylate Synthase) sa cible principale. La surexpression de la TS est un biomarqueur de résistance au 5-FU utilisé en clinique. La TS a été montrée comme étant O-GlcNAcylée mais le rôle de cette MPT n’a pas été élucidé. Il nous est donc paru intéressant d’analyser le « cross-talk » entre O-GlcNAcylation et réponse au 5-FU dans le CCR dans l’hypothèse que la O-GlcNAcylation pourrait impacter la sensibilité au 5-FU en régulant sa cible TS. Un modèle murin in vivo de CCR humains et des cellules coliques non cancéreuses et cancéreuses ont été utilisés pour analyser l’effet du 5-FU sur la O-GlcNAcylation globale des protéines et réciproquement l’impact de la O-GlcNAcylation sur le niveau et l’activité de la TS, et la réponse au 5-FU. Nos données in vitro corroborent nos résultats in vivo et soutiennent que le 5-FU diminue la O-GlcNAcylation globale et que, réciproquement, la O-GlcNAcylation augmente le niveau de TS et sensibilise le CCR au 5-FU. Nous avons déchiffré le mécanisme moléculaire sous-jacent mettant en lumière le rôle de la O-GlcNAcylation dans la stabilisation de la TS et sa protection contre la dégradation protéasomale. Deux sites de O-GlcNAcylation de la TS ont été identifiés : la Thr251 à l’interface de dimérisation de l’enzyme et la Thr306 dans la séquence dégron carboxy-terminale connue pour contrôler sa dégradation. Ensemble nos résultats proposent une nouvelle stratégie thérapeutique combinant le 5-FU à un inhibiteur de l’OGA afin d’améliorer la réponse du CCR à la chimiothérapie à base de 5-FU.
... Modyfikacja nie zmieniała wartości K m , natomiast obniżała wartość V max katalizowanej przez ST reakcji [68]. Zastosowanie modelowania molekularnego pozwoliło ocenić mechanizm tego wpływu [69]. ...
Article
Full-text available
Thymidylate synthase ThyA (EC 2.1.1.45; encoded by the Tyms gene), having been for 60 years a molecular target in chemotherapy, catalyses the dUMP pyrimidine ring C(5) methylation reaction, encompassing a transfer of one-carbon group (the methylene one, thus at the formaldehyde oxidation level) from 6R-N5,10-methylenetetrahydrofolate, coupled with a reduction of this group to the methyl one, with concomitant generation of 7,8-dihydrofolate and thymidylate. New facts are presented, concerning (i) molecular mechanism of the catalyzed reaction, including the substrate selectivity mechanism, (ii) mechanism of inhibition by a particular inhibitor, N4-hydroxy-dCMP, (iii) structural properties of the enzyme, (iv) cellular localization, (v) potential posttranslational modifications of the enzyme protein and their influence on the catalytic properties and (vi) non-catalytic activities of the enzyme.
... The key binding site of TS has been reported [31,36] and involves amino acid residues of Phe225, Leu221 and Ile108. As reported, 5-FU was found to interact with two important amino acid residues of the active site (Leu221 and Phe225) [31]. ...
Article
Full-text available
Thymidylate synthase (TS), one of folate dependent enzymes, is a key and well-recognized target for anticancer agents. In this study, a series of 6-aryl-5- cyano thiouracil derivatives were designed and synthesized in accordance with essential pharmacophoric features of known TS inhibitors. Nineteen compounds were screened in vitro for their anti-proliferative activities toward HePG-2, MCF-7, HCT-116, and PC-3 cell lines. Compounds 21c, 21d, and 24 exhibited high anti-proliferative activity, comparable to that of 5-fluorouracil. Additionally, ten compounds with potent anti-proliferative activities were further evaluated for their ability to inhibit TS enzyme. Six compounds (21b, 21c, 21d, 22, 23 and 24) demonstrated potent dose-related TS inhibition with IC50 values ranging from 1.57 to 3.89μM. The in vitro TS activity results were consistent with those of the cytotoxicity assay where the most potent anti proliferative compounds of the series showed good TS inhibitory activity comparable to that of 5 fluorouracil. Furthermore, molecular docking studies were carried out to investigate the binding pattern of the designed compounds with the prospective target, TS (PDB-code: 1JU6).
... The key binding site of TS has been reported [31,36] and involves amino acid residues of Phe225, Leu221 and Ile108. As reported, 5-FU was found to interact with two important amino acid residues of the active site (Leu221 and Phe225) [31]. ...
... Rights reserved www.nature.com/scientificreports/ minimum frequency value 45 , or the probability of the most likely conformation, and the number of minima over a case dependent threshold of frequency 46 . The conformational transitions of the E25 are reflected in different regions of the free energy landscape (FEL), which is depicted on the basis of the projection of the first two principal components of the Cα trajectory ( Fig. 6(a) and (b)). ...
Article
Full-text available
ADP-ribosylhydrolases (ARH1, ARH2 and ARH3) are a family of enzymes to catalyze ADP-ribosylation, a reversible and covalent post-translational modification (PTM). There are four phosphorylated sites (Tyr-4, Tyr-19, Tyr-20, and Tyr-205) in ARH1. To explore the structural changes and functional impact induced by phosphorylation, molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed for the phosphorylated and non-phosphorylated ARH1 with the ligands. MD simulations results indicate that: (1) Glu-25 is more frequently in the α helix group in the phosphorylated state with the adenosine-5-diphosphate-ribosylarginine (ADP-RA) complex (51.56%) than that of the non-phosphorylated state(2.12%); (2) Ser-124 and Ser-264 become less flexible in the phosphorylated state with ADP-RA complex, which helps two residues form hydrogen bonds with ADP-RA; and (3) Tyr-211 is also less flexible in the phosphorylated state with ADP-RA complex, which helps stabilize the cation-π interaction of Y211-R119. All these changes facilitate ADP-RA to bind ARH1. In addition, according to the crystal structure of adenosine-5-diphosphate-ribose (ADP-ribose) in complex with non-phosphorylated and phosphorylated ARH1, the possible unbinding pathways of ADP-ribose from non-phosphorylated and phosphorylated ARH1 were explored respectively using SMD simulations. Our results show that phosphorylated ARH1 has more ordered structures than the non-phosphorylated type.
... It was reported that hsTSase could under particular circumstances have methylated glutamates and phosphorylation at serine. However, neither the modification sites nor the functional effect of those modifications, nor the conditions under which these forms are generated, have been unequivocally documented [14,16,17]. While a plethora of kinetic, structural and mechanistic studies are available on bacterial TSase [1,4,8,[18][19][20][21][22], mammalian TSase lacks such deep interrogations. ...
Article
Full-text available
Thymidylate Synthase (TSase) is a highly conserved enzyme that catalyzes the production of the DNA building block thymidylate. Structurally, functionally and mechanistically, bacterial and mammalian TSases share remarkable similarities. Because of this closeness, bacterial enzymes have long been used as model systems for human TSase. Furthermore, while TSase inhibitors have long served as chemotherapeutic drugs, no TSase inhibitor serves as an antibiotic. Despite their high resemblance, the mammalian TSases are distinct in a few known aspects, such as having a N-terminal tail and two insertions in the primary sequence and active/inactive conformations. Here, we aim to comprehensively characterize human (hs) TSase and delineate its contrasts and the similarities to the well-studied Escherichia coli (ec) TSase. We found that, in contrast to ecTSase, Mg²⁺ does not enhance reaction rates for hsTSase. The temperature dependence of intrinsic kinetic isotope effects (KIEs), on the other hand, suggests that Mg²⁺ has little or no impact on the transition state of hydride transfer in either enzyme, and that the transition state for the hydride transfer in hsTSase is looser than in ecTSase. Additionally, the substrates’ binding order is strictly ordered for ecTSase but slightly less ordered for hsTSase. The observed kinetic and functional differences between bacterial and human enzymes may aid in the development of antibiotic drugs with reduced toxicity.
... catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by N 5,10 -methylenetetrahydrofolate (meTHF) to generate thymidylate (dTMP) and dihydrofolate. Being the sole de novo source of dTMP in animal cells [1], the enzyme is consequently a target of antitumor, antiviral, antifungal, and antiprotozoan chemotherapy [2][3][4][5][6][7][8][9]. ...
Article
Full-text available
With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.
Article
Extracellular signal-regulated kinase 2 (ERK2) is a promising target for designs and development of anticancer drugs. Molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann method were applied to study binding difference of ADP and ATP to ERK2. The results prove that the binding ability of ATP to ERK2 is stronger than that of ADP. Principal component analysis performed by using MD trajectories suggest that bindings of ADP and ATP to ERK2 change motion directions of two helixes α1 and α2. Residue-based free energy decomposition method was adopted to calculate contributions of separate residues to associations of ADP and ATP with ERK2. The results show that ADP and ATP produce strong CH-π interactions with five residues Ile29, Val37, Ala50, Leu105 and Leu154. In addition, five hydrogen bonding interactions of ADP and ATP with residues Lys52, Gln103, Asp104 and Met106 also stabilize bindings of ADP and ATP to ERK2. Overall, the CH-π interactions of ATP with five residues Ile29, Val37, Ala50, Leu105 and Leu154 are stronger than ADP. This study is expected to contribute a significant theoretical hints for designs of anti-cancer drugs targeting ERK2. This article is protected by copyright. All rights reserved.
Article
Full-text available
Post-translational phosphorylation is a ubiquitous mechanism for cellular regulation, playing a crucial role in a diverse array of processes. At the molecular level, the phosphorylation seems to cause electrostatic perturbations which modulate the energy landscape governing protein folding, activity, protein-protein interactions and, conformational dynamics. However, details on structural effects induced by phosphorylation of proteins are still poorly understood. In this context, computational simulations recently provided a valuable alternative in the elucidation of principles and mechanisms of protein phosphorylation. In the present contribution, we review several recent studies devoted to molecular dynamics simulations of phospho-proteins with particular attention to proteins involved in the p53 pathway, ubiquitin pathway, protein kinases and intrinsically disordered proteins. Since application of biomolecular simulations to the investigation of mechanisms related to protein phospho-regulation is a newborn field for computational biology, our contribution could provide a suitable framework to plan future researches and to rationalize the available data in the field.
Article
Crystal structure is presented of the binary complex between potassium phosphoramidate-phosphorylated recombinant C. elegans thymidylate synthase and dUMP. On each monomer a single phosphoserine residue (Ser127) was identified, instead of expected phosphohistidine. As 31P NMR studies of both the phosphorylated protein and of potassium phosphoramidate potential to phosphorylate different amino acids, point to histidine as the only possible site of the modification, thermodynamically favored intermolecular phosphotransfer from histidine to serine is suggested.
Article
Full-text available
To develop alkyl halides for a promising prodrug system, a 5-fluorouracil prodrug containing a bromoethyl group (5-FUBr) was synthesized and its hydrophobicity, cytotoxicity and DNA-bonding ability were investigated in detail. Compare with 5-fluorouracil, 5-FUBr exhibits a great advantage of hydrophobicity and shows significant reduction in toxic side effect. To explore the mechanism of action of 5-FUBr at the molecular level, X-ray crystallography and molecular docking were exploited to make a more detailed analysis of the bromoethyl contribution to the construction of meaningful structure–activity relationship. Details of X-ray crystal structure of 5-FUBr suggest that 5-fluorouracil may be more apt to be released from 5-FUBr. The appearance of the bromoethyl group in 5-FUBr makes a remarkable impact on inhibition of thymidylate synthase (TS), and the impact of subtle structural variation between 5-fluorouracil and 5-FUBr should be taken into account in the process of developing this family of 5-fluorouracil prodrugs.
Article
Fibrillation of β-amyloid is recognized as a key process leading to the development of Alzheimer's disease. Small peptides called β-sheet breakers were found to inhibit the process of β-amyloid fibrillation and to dissolve amyloid fibrils in vitro, in vivo, and in cell culture studies . The mechanism by which peptide inhibition takes place remains elusive and a detailed model needs to be established. Here, we present new insights into the possible role of consecutive Phe residues, present in the structure of β-sheet breakers, supported by the results obtained by means of MD simulations. We performed a 30-ns MD of two β-sheet breakers: iAβ5 (LPFFD) and iAβ6 (LPFFFD) which have two and three consecutive Phe residues, respectively. We have found that Phe rings in these peptides tend to form stacked conformations. For one of the peptides - iAβ6 - the calculated electrostatic contribution to free energy of one of the conformers with three rings stacked (c2) is significantly lower than that corresponding to the unstacked one (c1), two rings stacked (c0) and second conformer with three rings stacked (c3). This may favor the interaction of the c2 conformer with the target on amyloid fibril. We hypothesize that the mechanism of inhibition of amyloidogenesis by β-sheet breaker involves competition among π-stacked Phe residues of the inhibitor and π-stacking within the β-amyloid fibril. iAβ6 may be a promising candidate for a lead compound of amyloidogenesis inhibitors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Article
Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (K d below 0.5 μM, k on = 10.31 × 10 ⁴ M ⁻¹ s ⁻¹ and k off = 1.40 × 10 ⁻³ s ⁻¹ ) and calculated Gibbs free energy (−36.4 kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level.
Article
Peroxiredoxin 6 (PRDX6), a 1-Cys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post-translational modifications (PTMs) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTMs in PRDX6 utilizing proteomic tools including nanoUPLC-ESI-q-TOF MS/MS employing selectively excluded mass screening analysis (SEMSA) in conjunction with MOD(i) and MODmap algorithm. We chose PRDX6 obtained from liver tissues from two inbred mouse strains, C57BL/6J and C3H/HeJ, which vary in their susceptibility to high-fat diet-induced obesity and atherosclerosis, and a B16F10 melanoma cell line for this study. When PRDX6 protein samples were separated on 2D-PAGE based on pI, several PRDX6 spots appeared. They were purified and the low abundant PTMs in each PRDX6 spot were analyzed. Unexpected mass shifts (Δm = -34, +25, +64, +87, +103, +134, +150, +284 Da) observed at active site cysteine residue (Cys47) were quantified using precursor ion intensities. Mass differences of -34, +25, and +64 Da are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and Cys-SO(2) -SH, respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da) as well as unknown modifications (+134, +150, +284 Da). Comprehensive analysis of these PTMs revealed that the PRDX6 exists as a heterogeneous mixture of molecules containing a multitude of PTMs. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX6 from mouse liver tissues included, among others, mono- and dioxidation at Trp and Met, acetylation at Lys, and deamidation at Asn and Gln. Comprehensive identification of the diverse PTMs occurring in this bifunctional PRDX6 enzyme should help understand how PRDX6 plays key roles in oxidative stresses.
Article
Full-text available
Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
Article
In this study, a series of thiouracil derivatives were designed and synthesized based on conventional approach and pharmacophoric features essential for TS inhibitors. The chemical structures of all synthesized compounds were elucidated by various techniques ranging from micro-elemental analyses to spectral analyses. The new thiouracil derivatives were evaluated for their anticancer and antibacterial activities. All the new synthesized compounds were evaluated in vitro against MCF-7 cell line. The anticancer results displayed that compounds 8, 11, 13a, and 12, exhibit the highly significant effect against breast cancer cell line with IC50 values of 3.80, 4.00, 4.50, and 4.70 µg/ml, respectively compared with doxorubicin. Furthermore, molecular docking studies were performed to suggest possible mechanism of action of the designed compounds and explain the anti-breast cancer results with prospective target, thymidylate synthase (PDB:1JU6). On the other hand, the antibacterial activity of the new compounds was screened against three significant representative strains including Escherichia coli, and Pseudomonas aeruginosa as gram negative bacterium and Staphylococcus aureus as gram positive bacterium using agar well diffusion method. The antibacterial activity results revealed that most of the tested compounds exhibited significant antibacterial activity. In particularly, compounds 13a, and 13b were found to be the most potent antibacterial agent with inhibition zone values of 38 and 35 mm at the concentration of 50 µg/ml against Escherichia coli, and inhibition zone values of 25 and 23 mm at the concentration of 50 µg/ml against Staphylococcus aureus. However, all tested strains showed resistance to synthesized compounds except, compound 7 which exhibited significant activity only against Pseudomonas aeruginosa with inhibition zone values of 22 mm at the concentration of 50 µg/ml. Further, molecular docking investigation was carried out to gain insight into the binding mode of the most promising compounds using crystal structure of S. aureus DNA gyrase complex with ciprofloxacin (PDB ID: 2XCT).
Article
A series of novel N’-(2-thiouracil-5-oyl)hydrazones were designed and chemically synthesized using an active substructure combination method. The synthesized compounds were structurally characterized on the basis of elemental (%) analyses, IR, MS, ¹H NMR, and ¹³C NMR spectra. All the prepared compounds were evaluated in vitro against MCF-7 human breast cell line using MTT assay. The anticancer results showed that compounds 3j, 4a, 3c, 3b, and 3h exhibit the most prominent effect against breast cancer cell line with IC50 values of 3.40, 3.50, 3.60, 3.70, and 3.80 µg/ml, respectively using doxorubicin as a control drug. Moreover, molecular docking studies were also performed in order to identify the binding mode mechanism of these compounds with prospective target, thymidylate synthase (PDB:1JU6). On the other hand, the antibacterial activities of all prepared compounds were screened in vitro against three bacterial strains, namely, Escherichia coli, and Pseudomonas aeruginosa as Gram negative bacteria and Staphylococcus aureus as a Gram positive bacterium using agar well diffusion method. The antibacterial activity results revealed that most of the compounds under test were inactive however, among all the tested compounds, only 3g and 4a, in a concentration of 50 µg/ml showed a high antibacterial activity against the three used bacterial strained. It is worthy to note that the potency of 3g against Escherichia coli was comparable to that of the reference drug, while compound 2 exhibited significant activity against Pseudomonas aeruginosa only and compound 3j displayed low activity against Staphylococcus aureus. The structure-antibacterial activity relationship analysis can be modulated by the presence of aromatic or heteroaromatic moiety containing more lipophilic character significantly contributed to antibacterial activity. In addition, the drug-likeness properties have predicted for the target compounds.
Article
Substrate specificity of protein kinases is of fundamental importance for the integrity and fidelity of signaling pathways. Glycogen synthase kinase 3β (GSK3β) has a unique substrate specificity that prefers phosphorylation of its substrates at the P+4 serine before it can further phosphorylate the substrate at the P0 serine in the canonical motif SXXXS(p), where S(p) is the primed phosphorylation site. The detailed phosphorylation mechanism, however, is not clearly understood. In this study, a three-dimensional (3D) model of the ternary complex of GSK3β, ATP, and the phosphorylated glycogen synthase (pGS), termed GSK3β/ATP/pGS, is constructed using a hierarchical approach and by integrating molecular modeling and molecular dynamics (MD) simulations. Based on the 3D model, the substrate primed phosphorylation mechanism is investigated via two 12 ns comparative MD simulations of the GSK3β/ATP/pGS and GSK3β/ATP/GS systems, which differ in the phosphate group bound to the P+4 serine of GS. In agreement with structural analysis, computed binding free energies reveal that the binding of pGS to GSK3β is favored in the prephosphorylated state compared with the GS native state. More importantly, comparison with the system simulated without primed phosphorylation in the GSK3β/ATP/GS complex shows that for an optimal phosphorylation reaction to occur, the pGS priming phosphate in the GSK3β/ATP/pGS system optimizes the proper orientation of the GSK3β N- and C-terminal domains and clamps the P0 serine of pGS in the appropriate configuration for interaction with the ATP γ-phosphate within the catalytic groove.
Article
Full-text available
We present a simple method for compression and management of very large molecular dynamics trajectories. The approach is based on the projection of the Cartesian snapshots collected along the trajectory into an orthogonal space defined by the eigenvectors obtained by diagonalization of the covariance matrix. The transformation is mathematically exact when the number of eigenvectors equals 3N−6 (N being the number of atoms), and in practice very accurate even when the number of eigenvectors is much smaller, permitting a dramatic reduction in the size of trajectory files. In addition, we have examined the ability of the method, when combined with interpolation, to recover dense samplings (snapshots collected at a high frequency) from more sparse (lower frequency) data as a method for further data compression. Finally, we have investigated the possibility of using the approach when extrapolating the behavior of the system to times longer than the original simulation period. Overall our results suggest that the method is an attractive alternative to current approaches for including dynamic information in static structure files such as those deposited in the Protein Data Bank.
Article
Full-text available
A historical perspective on the application of molecular dynamics (MD) to biological macromolecules is presented. Recent developments combining state-of-the-art force fields with continuum solvation calculations have allowed us to reach the fourth era of MD applications in which one can often derive both accurate structure and accurate relative free energies from molecular dynamics trajectories. We illustrate such applications on nucleic acid duplexes, RNA hairpins, protein folding trajectories, and protein−ligand, protein−protein, and protein−nucleic acid interactions.
Article
Full-text available
The previously developed particle mesh Ewald method is reformulated in terms of efficient B‐spline interpolation of the structure factors. This reformulation allows a natural extension of the method to potentials of the form 1/r p with p≥1. Furthermore, efficient calculation of the virial tensor follows. Use of B‐splines in place of Lagrange interpolation leads to analytic gradients as well as a significant improvement in the accuracy. We demonstrate that arbitrary accuracy can be achieved, independent of system size N, at a cost that scales as N log(N). For biomolecular systems with many thousands of atoms this method permits the use of Ewald summation at a computational cost comparable to that of a simple truncation method of 10 Å or less.
Article
Full-text available
A numerical algorithm integrating the 3N Cartesian equations of motion of a system of N points subject to holonomic constraints is formulated. The relations of constraint remain perfectly fulfilled at each step of the trajectory despite the approximate character of numerical integration. The method is applied to a molecular dynamics simulation of a liquid of 64 n-butane molecules and compared to a simulation using generalized coordinates. The method should be useful for molecular dynamics calculations on large molecules with internal degrees of freedom.
Article
Full-text available
In molecular dynamics (MD) simulations the need often arises to maintain such parameters as temperature or pressure rather than energy and volume, or to impose gradients for studying transport properties in nonequilibrium MD. A method is described to realize coupling to an external bath with constant temperature or pressure with adjustable time constants for the coupling. The method is easily extendable to other variables and to gradients, and can be applied also to polyatomic molecules involving internal constraints. The influence of coupling time constants on dynamical variables is evaluated. A leap‐frog algorithm is presented for the general case involving constraints with coupling to both a constant temperature and a constant pressure bath.
Article
Full-text available
Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.
Article
Full-text available
Molecular mechanics models have been applied extensively to study the dynamics of proteins and nucleic acids. Here we report the development of a third-generation point-charge all-atom force field for proteins. Following the earlier approach of Cornell et al., the charge set was obtained by fitting to the electrostatic potentials of dipeptides calculated using B3LYP/cc-pVTZ//HF/6-31G** quantum mechanical methods. The main-chain torsion parameters were obtained by fitting to the energy profiles of Ace-Ala-Nme and Ace-Gly-Nme di-peptides calculated using MP2/cc-pVTZ//HF/6-31G** quantum mechanical methods. All other parameters were taken from the existing AMBER data base. The major departure from previous force fields is that all quantum mechanical calculations were done in the condensed phase with continuum solvent models and an effective dielectric constant of epsilon = 4. We anticipate that this force field parameter set will address certain critical short comings of previous force fields in condensed-phase simulations of proteins. Initial tests on peptides demonstrated a high-degree of similarity between the calculated and the statistically measured Ramanchandran maps for both Ace-Gly-Nme and Ace-Ala-Nme di-peptides. Some highlights of our results include (1) well-preserved balance between the extended and helical region distributions, and (2) favorable type-II poly-proline helical region in agreement with recent experiments. Backward compatibility between the new and Cornell et al. charge sets, as judged by overall agreement between dipole moments, allows a smooth transition to the new force field in the area of ligand-binding calculations. Test simulations on a large set of proteins are also discussed.
Article
Full-text available
Thymidylate synthase (TS) is a well-validated target for cancer chemotherapy. TS was established as the principal target of the widely used anticancer drug 5-fluorouracil (5FU). The 5FU metabolite FdUMP forms a covalent complex with TS that is stabilized by 5-formyl tetrahydrofolate (leucovorin; LV). Numerous chemical strategies have been employed to develop novel TS inhibitors that are superior to 5FU/LV. 5FU is non-ideal as a TS-inhibitory drug because it is only inefficiently converted to FdUMP, while the remainder of the administered dose is converted to toxic metabolites. My laboratory has explored the utility of FdUMP[N] compounds (oligodeoxynucleotides comprised of FdUMP nucleotides) as FdUMP pro-drugs. FdUMP[N] compounds result in potent TS-inhibition, and display many advantages relative to 5FU/LV. A number of other chemical strategies have also been employed to develop pro-drugs, or metabolic precursors of FdUMP, and several of these strategies will be reviewed. In addition to chemical strategies to develop FdUMP pro-drugs, a number of chemical strategies have been devised to develop molecules that resemble the reduced folate co-factor required for TS catalysis. The synthesis of antifolates that have TS-inhibitory activity, such as Raltitrexed, has resulted in compounds that are effective and specific TS-inhibitors and, in some cases, have clinical potential. Chemical strategies that target TS mRNA for destruction are also being explored as potential chemotherapeutics. These diverse chemical approaches to control TS activity in tumor cells for the treatment of cancer will be reviewed.
Article
Full-text available
Recent methodologies applied to the drug discovery process, such as genomics and proteomics, have greatly implemented our basic understanding of drug action and are giving more input to medicinal chemists, in finding genuinely new targets and opportunities for the development of drugs with original mechanisms of action. In this paper, an example of the successful application of some new techniques to the target enzymes with the Thymidylate Synthase (TS) function is given. The improved knowledge of the complex mechanism of the biological pathways in which thymidylate synthase is involved represents a unique chance to find new mechanism-based inhibitors, aimed to treat not only cancerous diseases, but also infectious pathologies. Thymidylate synthase (TS or ThyA) has long been considered as one of the best-known drug targets in the anti-cancer area, after which old and new drugs, such as 5-fluoro uracil and the anti-folate ZD1694, have been introduced into chemotherapy to treat solid tumours. Only a few attempts have been made to find non-classical anti-folate inhibitors that are dissimilar to the folate co-factor, with the aim of finding unshared protein target domains on the enzyme structure, in order to specifically inhibit TS enzymes from pathogens. Only recently from omic studies, a new Thymidylate Synthase Complementing Protein (TSCP or ThyX) has been identified in a number of pathogens, showing a different structure with respect to human TS, thus opening new avenues to specific inhibitions. A depiction of the most recent progress in the study of Thymidylate Synthase enzymes is presented in the following sections.
Article
A fast analytical formula was derived for the calculation of approximate atomic and molecular van der Waals (vdWSA), and solvent-accessible surface areas (SASAs), as well as the first and second derivatives of these quantities with respect to atomic coordinates. This method makes use of linear combinations of terms composed from pairwise overlaps of hard spheres; therefore, we term this the LCPO method for linear combination of pairwise overlaps. For higher performance, neighbor-list reduction (NLR) was applied as a preprocessing step. Eighteen compounds of different sizes (8–2366 atoms) and classes (organic, proteins, DNA, and various complexes) were chosen as representative test cases. LCPO/NLR computed the SASA and first derivatives of penicillopepsin, a protein with 2366 atoms, in 0.87 s (0.22 s for the creation of the neighbor list, 0.35 s for NLR, and 0.30 s for SASA and first derivatives) on an SGI R10000/194 Mhz processor. This appears comparable to or better than timings reported previously for other algorithms. The vdWSAs were in good agreement with the numerical results: relative errors for total molecular surface areas ranged from 0.1 to 2.0% and average absolute atomic surface area deviations from 0.3 to 0.7 Ų. For SASAs without NLR, the LCPO method exhibited relative errors in the range of 0.4–9.2% for total molecular surface areas and average absolute atomic surface area deviations of 2.0–2.7 Ų; with NLR the relative molecular errors ranged from 0.1 to 7.8% and the average absolute atomic surface area deviation from 1.6 to 3.0 Ų. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 217–230, 1999
Article
The LIGPLOT program automatically generates schematic 2-D representations of protein-ligand complexes from standard Protein Data Bank file input. The output is a colour, or black-and-white, PostScript file giving a simple and informative representation of the intermolecular interactions and their strengths, including hydrogen bonds, hydrophobic interactions and atom accessibilities. The program is completely general for any ligand and can also be used to show other types of interaction in proteins and nucleic acids. It was designed to facilitate the rapid inspection of many enzyme complexes, but has found many other applications.
Article
Monte Carlo statistical mechanics simulations have been carried out for a dilute solution of n‐butane in water at 25 °C and 1 atm. The intermolecular interactions were described by Coulomb and Lennard‐Jones terms in the TIPS format including the TIPS2 parameters for water. The internal rotation about the central CC bond in n‐butane was included using a rotational potential based on molecular mechanics (MM2) calculations. The precision of the simulation results was enhanced by preferential sampling and by umbrella sampling for the internal rotation over chopped barriers. Conformational results are also reported from a long Monte Carlo run for pure liquid n‐butane using umbrella sampling. Although no condensed phase effect is found on the conformational equilibrium in pure liquid n‐butane, there is a pronounced increase in the gauche population of n‐butane upon transfer from the gas phase to aqueous solution. The latter finding is in near quantitative accord with the shift predicted by Pratt and Chandler from their theoretical model. It is also consistent with the basic tenet of the hydrophobic effect regarding the folding of natural and synthetic polymers in water. In addition, detailed structural results for the system are reported. Notably, the water molecules in the first shell around n‐butane have normal bonding energies and hydrogen bonding profiles for bulk water. However, since their coordination numbers are low, this situation can only be achieved by greater ordering which is entropically costly. The validity of the observations is supported by the computed heat and volume of solution which are in accord with experimental data.
Article
We present a new approach to generating electrostatic potential (ESP) derived charges for molecules. The major strength of electrostatic potential derived charges is that they optimally reproduce the intermolecular interaction properties of molecules with a simple two-body additive potential, provided, of course, that a suitably accurate level of quantum mechanical calculation is used to derive the ESP around the molecule. Previously, the major weaknesses of these charges have been that they were not easily transferable between common functional groups in related molecules, they have often been conformationally dependent, and the large charges that frequently occur can be problematic for simulating intramolecular interactions. Introducing restraints in the form of a penalty function into the fitting process considerably reduces the above problems, with only a minor decrease in the quality of the fit to the quantum mechanical ESP. Several other refinements in addition to the restrained electrostatic potential (RESP) fit yield a general and algorithmic charge fitting procedure for generating atom-centered point charges. This approach can thus be recommended for general use in molecular mechanics, molecular dynamics, and free energy calculations for any organic or bioorganic system.
Article
A generalized Born (GB) model has been applied to molecular dynamics simulations of the A- and B-forms of a duplex DNA d(CCAACGTTGG)2 and the corresponding duplex RNA r(CCAACGUUGG)2, resulting in good agreement with simulations using explicit water solvent in terms of both structure and energetics. In particular, the A → B energy differences derived from GB trajectories for both DNA and RNA closely match those obtained earlier using explicit water simulations and finite-difference Poisson−Boltzmann calculations. A GB simulation starting from A-form DNA converges to B-DNA within 20 ps, more than 20 times faster than the transition from A- to B-DNA in explicit solvent simulations. For B-form d(CCAACGTTGG)2, fluctuations about the mean are highly correlated between GB and explicit water simulations, being slightly larger in the former, and the essential subspaces found from principal component analysis overlap to a high degree. Hence, for many purposes this parametrization offers an alternative to more expensive explicit water simulations for studies of nucleic acid energetics and structure.
Article
We present the derivation of charges of ribo- and deoxynucleosides, nucleotides, and peptide fragments using electrostatic potentials obtained from ab initio calculations with the 6-31G* basis set. For the nucleic acid fragments, we used electrostatic potentials of the four deoxyribonucleosides (A, G, C, T) and four ribonucleosides (A, G, C, U) and dimethylphosphate. The charges for the deoxyribose nucleosides and nucleotides are derived using multiple-molecule fitting and restrained electrostatic potential (RESP) fits,1,2 with Lagrangian multipliers ensuring a net charge of 0 or ± 1. We suggest that the preferred approach for deriving charges for nucleosides and nucleotides involves allowing only C1′ and H1′ of the sugar to vary as the nucleic acid base, with the remainder of sugar and backbone atoms forced to be equivalent. For peptide fragments, we have combined multiple conformation fitting, previously employed by Williams3 and Reynolds et al.,4 with the RESP approach1,2 to derive charges for blocked dipeptides appropriate for each of the 20 naturally occuring amino acids. Based on our results for propyl amine,1,2 we suggest that two conformations for each peptide suffice to give charges that represent well the conformationally dependent electrostatic properties of molecules, provided that these two conformations contain different values of the dihedral angles that terminate in heteroatoms or hydrogens attached to heteroatoms. In these blocked dipeptide models, it is useful to require equivalent N—H and CO charges for all amino acids with a given net charge (except proline), and this is accomplished in a straightforward fashion with multiple-molecule fitting. Finally, the application of multiple Lagrangian constraints allows for the derivation of monomeric residues with the appropriate net charge from a chemically blocked version of the residue. The multiple Lagrange constraints also enable charges from two or more molecules to be spliced together in a well-defined fashion. Thus, the combined use of multiple molecules, multiple conformations, multiple Lagrangian constraints, and RESP fitting is shown to be a powerful approach to deriving electrostatic charges for biopolymers. © 1995 John Wiley & Sons, Inc.
Article
Generalized Born (GB) models provide an attractive way to include some thermodynamic aspects of aqueous solvation into simulations that do not explicitly model the solvent molecules. Here we discuss our recent experience with this model, presenting in detail the way it is implemented and parallelized in the AMBER molecular modeling code. We compare results using the GB model (or GB plus a surface-area based “hydrophobic” term) to explicit solvent simulations for a 10 base-pair DNA oligomer, and for the 108-residue protein thioredoxin. A slight modification of our earlier suggested parameters makes the GB results more like those found in explicit solvent, primarily by slightly increasing the strength of NHO and NHN internal hydrogen bonds. Timing and energy stability results are reported, with an eye toward using these model for simulations of larger macromolecular systems and longer time scales. © 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 56: 275–291, 2001
Article
Simulations of the HIV‐1 protease unit cell using a 9 Å cutoff, 9/18 Å ‘‘twin‐range’’ cutoff, and full Ewald sums have been carried out to 300 ps. The results indicate that long‐range electrostaticinteractions are essential for proper representation of the HIV‐1 protease crystal structure. The 9 Å simulation did not converge in 300 ps. Inclusion of a 9/18 Å ‘‘twin‐range’’ cutoff showed significant improvement. Simulation using the Ewald summation convention gave the best overall agreement with x‐ray crystallographic data, and showed the least internal differences in the time average structures of the asymmetric units. The Ewald simulation represents an efficient implementation of the Particle Mesh Ewald method [Darden et al., J. Chem. Phys. 98, 10 089 (1993)], and illustrates the importance of including long‐range electrostatic forces in large macromolecular systems.
Article
A fast analytical formula was derived for the calculation of approximate atomic and molecular van der Waals (vdWSA), and solvent-accessible surface areas (SASAs), as well as the first and second derivatives of these quantities with respect to atomic coordinates. This method makes use of linear combinations of terms composed from pairwise overlaps of hard spheres; therefore, we term this the LCPO method for linear combination of pairwise overlaps. For higher performance, neighbor-list reduction (NLR) was applied as a preprocessing step. Eighteen compounds of different sizes (8–2366 atoms) and classes (organic, proteins, DNA, and various complexes) were chosen as representative test cases. LCPO/NLR computed the SASA and first derivatives of penicillopepsin, a protein with 2366 atoms, in 0.87 s (0.22 s for the creation of the neighbor list, 0.35 s for NLR, and 0.30 s for SASA and first derivatives) on an SGI R10000/194 Mhz processor. This appears comparable to or better than timings reported previously for other algorithms. The vdWSAs were in good agreement with the numerical results: relative errors for total molecular surface areas ranged from 0.1 to 2.0% and average absolute atomic surface area deviations from 0.3 to 0.7 Å2. For SASAs without NLR, the LCPO method exhibited relative errors in the range of 0.4–9.2% for total molecular surface areas and average absolute atomic surface area deviations of 2.0–2.7 Å2; with NLR the relative molecular errors ranged from 0.1 to 7.8% and the average absolute atomic surface area deviation from 1.6 to 3.0 Å2. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 217–230, 1999
Article
Thymidylate synthase (TS) was found to be a substrate for both catalytic subunits of human CK2, with phosphorylation by CK2alpha and CK2alpha' characterized by similar K(m) values, 4.6microM and 4.2microM, respectively, but different efficiencies, the apparent turnover number with CK2alpha being 10-fold higher. With both catalytic subunits, phosphorylation of human TS, like calmodulin and BID, was strongly inhibited in the presence of the regulatory subunit CK2beta, the holoenzyme being activated by polylysine. Phosphorylation of recombinant human, rat, mouse and Trichinella spiralis TSs proteins was compared, with the human enzyme being apparently a much better substrate than the others. Following hydrolysis and TLC, phosphoserine was detected in human and rat, and phosphotyrosine in T. spiralis, TS, used as substrates for CK2alpha. MALDI-TOF MS analysis led to identification of phosphorylated Ser(124) in human TS, within a sequence LGFS(124)TREEGD, atypical for a CK2 substrate recognition site. The phosphorylation site is located in a region considered important for the catalytic mechanism or regulation of human TS, corresponding to the loop 107-128. Following phosphorylation by CK2alpha, resulting in incorporation of 0.4mol of phosphate per mol of dimeric TS, human TS exhibits unaltered K(m) values for dUMP and N(5,10)-methylenetetrahydrofolate, but a 50% lower turnover number, pointing to a strong influence of Ser(124) phosphorylation on its catalytic efficiency.
Article
The LIGPLOT program automatically generates schematic 2-D representations of protein-ligand complexes from standard Protein Data Bank file input The output is a colour, or black-and-white, PostScript file giving a simple and informative representation of the intermolecular interactions and their strengths, including hydrogen bonds, hydrophobic interactions and atom accessibilities. The program is completely general for any ligand and can also be used to show other types of interaction in proteins and nucleic acids. It was designed to facilitate the rapid inspection of many enzyme complexes, but has found many other applications
Article
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.
Article
Analysis of extended molecular dynamics (MD) simulations of lysozyme in vacuo and in aqueous solution reveals that it is possible to separate the configurational space into two subspaces: (1) an "essential" subspace containing only a few degrees of freedom in which anharmonic motion occurs that comprises most of the positional fluctuations; and (2) the remaining space in which the motion has a narrow Gaussian distribution and which can be considered as "physically constrained." If overall translation and rotation are eliminated, the two spaces can be constructed by a simple linear transformation in Cartesian coordinate space, which remains valid over several hundred picoseconds. The transformation follows from the covariance matrix of the positional deviations. The essential degrees of freedom seem to describe motions which are relevant for the function of the protein, while the physically constrained subspace merely describes irrelevant local fluctuations. The near-constraint behavior of the latter subspace allows the separation of equations of motion and promises the possibility of investigating independently the essential space and performing dynamic simulations only in this reduced space.
Article
Thymidylate synthase undergoes a major conformational change upon ligand binding, where the carboxyl terminus displays the largest movement (approximately 4 A). This movement from an "open" unliganded state to the "closed" complexed conformation plays a crucial role in the correct orientation of substrates and in product formation. The mutant lacking the C-terminal valine (V316Am) of the enzyme is inactive. X-ray crystal structures of V316Am and its complexes with dUMP, FdUMP, and both FdUMP and CH2H4folate are described. The structures show that ligands are bound within the active site, but in different modes than those in analogous, wild-type thymidylate synthase structures. The 2.7-A binary complex structures of V316Am with FdUMP and dUMP show that the pyrimidine and ribose moieties of the nucleotides are pivoted approximately 20 degrees around the 3'-hydroxyl compared to dUMP in the wild-type enzyme. The 2.7-A crystal structure of V316Am complexed with cofactor, CH2H4folate, and the substrate analog, FdUMP, shows these ligands bound in an open conformation similar to that of the unliganded enzyme. In this ternary complex, the imidazolidine ring of the cofactor is open and has reacted with water to form 5-HOCH2H4folate. 5-HOCH2H4folate is structural evidence for the 5-iminium ion intermediate, which is the proposed reactive form of CH2H4folate. The altered ligand binding modes observed in the three V316Am complex structures open new venues for the design of novel TS inhibitors.
Article
In thymidylate synthase (TS, EC 2.1.1.45), the only side chain in direct hydrogen bonding with the pyrimidine ring of the substrate dUMP is asparagine 229 (N229). In binary and ternary complexes, the carboxamide moiety of the side chain of N229 forms a cyclic hydrogen bond network bridging N-3 and O-4 of the uracil heterocycle. Most of the N229 mutants of TS bind dUMP and catalyze dTMP formation as well as the wild-type enzyme; thus, N229 does not contribute to binding of dUMP. Wild-type TS binds dCMP weakly and does not accept dCMP as a substrate. Mutations at N229 of TS modify the interaction of TS with dCMP. TS N229D and TS N229E catalyze the methylation of dCMP [Liu, L., & Santi, D. V. (1992) Biochemistry 31, 5010-5014]. With the exception of the TS N229Q, most of the N229 mutants bind dCMP as well as or tighter than dUMP and bind dCMP 300-3000-fold tighter than wild-type TS. We conclude that TS discriminates binding of dUMP versus dCMP by a 3-4 kcal mol-1 difference in binding energy by exclusion of dCMP from the active site. We propose that this exclusion is a consequence of untoward interactions between dCMP and the side-chain carboxamide group of the Asn or Gln at position 229 of TS. We speculate that exclusion of cytosine versus uracil by Asn or Gln may account for specificity observed in other protein-pyrimidine interactions.
Article
Thymidylate synthase (TS) methylates only dUMP, not dCMP. The crystal structure of TS.dCMP shows sCMP 4-NH2 excluded from the space between Asn-229 and His-199 by the hydrogen bonding and steric properties and Asn-229. Consequently, 6-C of dCMP is over 4 A from the active site sulfhydryl. The Asn-229 side chain is prevented from flipping 180 degrees to and orientation the could hydrogen bond to dCMP by a hydrogen bond network between conserved residues. Thus, the specific binding of dUMP by TS results from occlusion of competing substrates by steric and electronic effects of residues in the active site cavity. When Asn-229 is replaced by a cysteine, the Cys-229 S gamma rotates out of the active site, and the mutant enzyme binds both dCMP and dUMP tightly but does not methylate dCMP. Thus simply admitting dCMP into the dUMP binding site of TS is not sufficient for methylation of dCMP. Structures of nucleotide complexes of TS N229D provide a reasonable explanation for the preferential methylation of dCMP instead of dUMP by this mutant. In TS N229D.dCMP, Asp-229 forms hydrogen bonds to 3-N and 40NH2 of dCMP. Neither the Asp-229 carboxyl moiety nor ordered water appears to hydrogen bond to 4-O of dUMP. Hydrogen bonds to 4-O (or 4-NH2) have been proposed to stabilize reaction intermediates. If their absence in TS N229D.dUMP persists in the ternary complex, it could explain the 10(4)-fold decrease in kcat/Km for dUMP.
Article
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
Article
A method is presented for a more efficient sampling of the configurational space of proteins as compared to conventional sampling techniques such as molecular dynamics. The method is based on the large conformational changes in proteins revealed by the "essential dynamics" analysis. A form of constrained dynamics is performed, forcing the system to move along some of the essential coordinates. This results in a broader sampling of the essential subspace than in a comparable conventional molecular dynamics simulation without constraints. The new sampling method (essential dynamics sampling) was applied to the histidine-containing phosphocarrier protein HPr. The results indicate that the essential dynamics sampling method produces physically allowed structures, as estimated by the evaluation of many geometrical properties. In addition, a study of the motions in the essential subspace reveals a diffusion-like behavior.
Article
The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 A and 2.5 A resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 A away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten-fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.
Article
Studies from our laboratory have shown that the folate-dependent enzyme, thymidylate synthase (TS), functions as an RNA binding protein. There is evidence that TS, in addition to interacting with its own TS mRNA, forms a ribonucleoprotein complex with a number of other cellular mRNAs, including those corresponding to the p53 tumor suppressor gene and the myc family of transcription factors. Using both in vitro and in vivo model systems, we have demonstrated that the functional consequence of binding of TS protein to its own cognate mRNA, as well as binding of TS to the p53 mRNA, is translational repression. Herein, we review current work on the translational autoregulatory control of TS expression and discuss the molecular elements that are required for the TS protein-TS mRNA interaction. TS may play a critical role in regulating the cell cycle and the process of apoptosis through its regulatory effects on expression of p53 and perhaps other cell cycle related proteins. Finally, the ability of TS to function as a translational regulator may have important consequences with regard to the development of cellular resistance to various anticancer drugs.
Article
Protein kinase CK2 ('casein kinase II') has traditionally been classified as a messenger-independent protein serine/threonine kinase that is typically found in tetrameric complexes consisting of two catalytic (alpha and/or alpha') subunits and two regulatory beta subunits. Accumulated biochemical and genetic evidence indicates that CK2 has a vast array of candidate physiological targets and participates in a complex series of cellular functions, including the maintenance of cell viability. This review summarizes current knowledge of the structural and enzymic features of CK2, and discusses advances that challenge traditional views of this enzyme. For example, the recent demonstrations that individual CK2 subunits exist outside tetrameric complexes and that CK2 displays dual-specificity kinase activity raises new prospects for the precise elucidation of its regulation and cellular functions. This review also discusses a number of the mechanisms that contribute to the regulation of CK2 in cells, and will highlight emerging insights into the role of CK2 in cellular decisions of life and death. In this latter respect, recent evidence suggests that CK2 can exert an anti-apoptotic role by protecting regulatory proteins from caspase-mediated degradation. The mechanistic basis of the observation that CK2 is essential for viability may reside in part in this ability to protect cellular proteins from caspase action. Furthermore, this anti-apoptotic function of CK2 may contribute to its ability to participate in transformation and tumorigenesis.
Article
Thymidylate synthase (TS) is an E2F1-regulated enzyme that is essential for DNA synthesis and repair. TS protein and mRNA levels are elevated in many human cancers, and high TS levels have been correlated with poor prognosis in patients with colorectal, breast, cervical, bladder, kidney, and non-small cell lung cancers. In this study, we show that ectopic expression of catalytically active TS is sufficient to induce a transformed phenotype in mammalian cells as manifested by foci formation, anchorage independent growth, and tumor formation in nude mice. In contrast, comparable levels of two TS mutants carrying single point mutations within the catalytic domain had no transforming activity. In addition, we show that overexpression of TS results in apoptotic cell death following serum removal. These data demonstrate that TS exhibits oncogene-like activity and suggest a link between TS-regulated DNA synthesis and the induction of a neoplastic phenotype.
Article
Thymidylate synthase (TS) is an E2F1-regulated enzyme that is essential for DNA synthesis and repair. TS protein and mRNA levels are elevated in many human cancers, and high TS levels have been correlated with poor prognosis in patients with colorectal, breast, cervical, bladder, kidney, and non-small cell lung cancers. In this study, we show that ectopic expression of catalytically active TS is sufficient to induce a transformed phenotype in mammalian cells as manifested by foci formation, anchorage independent growth, and tumor formation in nude mice. In contrast, comparable levels of two TS mutants carrying single point mutations within the catalytic domain had no transforming activity. In addition, we show that overexpression of TS results in apoptotic cell death following serum removal. These data demonstrate that TS exhibits oncogene-like activity and suggest a link between TS-regulated DNA synthesis and the induction of a neoplastic phenotype.
Article
The free energy of binding between avidin and seven biotin analogues has been calculated with the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method. We have studied how the force field and the method to generate geometries affect the calculated binding free energies. Four different force fields were compared, but we saw no significant difference in the results. However, mixing the force fields used for the geometry generation and energy calculations is not recommended. In the molecular dynamics simulations, explicit water molecules must be used, but the size of the simulated system and the boundary conditions are less important. In fact, nonperiodic simulations with a fixed protein outside a relatively small simulated system (18 A) seem to be a proper approach. The mean absolute error was 9-19 kJ/mol, with a standard error of 5-15 kJ/mol, which arises mainly from the entropy term.
Article
The novel dioxatetraaza macrocycle [26]phen2N4O2, which incorporates two phenanthroline units, has been synthesized, and its acid-base behavior has been evaluated by potentiometric and 1H NMR methods. Six protonation constants were determined, and the protonation sequence was established by NMR. The location of the fifth proton on the phen nitrogen was confirmed by X-ray determinations of the crystal structures of the receptor as bromide and chloride salts. The two compounds have the general molecular formula {(H5[26]phen2N4O2)Xn(H2O)(5-n)}X(n-1) x mH2O, where X = Cl, n = 3, and m = 6 or X = Br, n = 4, and m = 5.5. In the solid state, the (H5[26]phen2N4O2)(5+) cation adopts a "horseshoe" topology with sufficient room to encapsulate three or four halogen anions through the several N-H...X hydrogen-bonding interactions. Two supermolecules {(H5[26]phen2N4O2)Xn(H2O)5-n}(5-n)(+) form an interpenetrating dimeric species, which was also found by ESI mass spectrum. Binding studies of the protonated macrocycle with aliphatic (ox(2-), mal(2-), suc(2-), cit(3-), cta(3-)) and aromatic (bzc(-), naphc(-), anthc(-), pyrc(-), ph(2-), iph(2-), tph(2-), btc(3-)) anions were determined in water by potentiometric methods. These studies were complemented by 1H NMR titrations in D2O of the receptor with selected anions. The Hi[26]phen2N4O2(i+) receptor can selectively uptake highly charged or extended aromatic carboxylate anions, such as btc(3-) and pyrc(-), in the pH ranges of 4.0-8.5 and <4.0, respectively, from aqueous solution that contain the remaining anions as pollutants or contaminants. To obtain further insight into these structural and experimental findings, molecular dynamics (MD) simulations were carried out in water solution.
Article
Guanine-rich DNA repeat sequences located at the terminal ends of chromosomal DNA can fold in a sequence-dependent manner into G-quadruplex structures, notably the terminal 150-200 nucleotides at the 3' end, which occur as a single-stranded DNA overhang. The crystal structures of quadruplexes with two and four human telomeric repeats show an all-parallel-stranded topology that is readily capable of forming extended stacks of such quadruplex structures, with external TTA loops positioned to potentially interact with other macromolecules. This study reports on possible arrangements for these quadruplex dimers and tetramers, which can be formed from 8 or 16 telomeric DNA repeats, and on a methodology for modeling their interactions with small molecules. A series of computational methods including molecular dynamics, free energy calculations, and principal components analysis have been used to characterize the properties of these higher-order G-quadruplex dimers and tetramers with parallel-stranded topology. The results confirm the stability of the central G-tetrads, the individual quadruplexes, and the resulting multimers. Principal components analysis has been carried out to highlight the dominant motions in these G-quadruplex dimer and multimer structures. The TTA loop is the most flexible part of the model and the overall multimer quadruplex becoming more stable with the addition of further G-tetrads. The addition of a ligand to the model confirms the hypothesis that flat planar chromophores stabilize G-quadruplex structures by making them less flexible.
  • F J Kaye
  • M Zajac-Kaye
Kaye, F. J.; Zajac-Kaye, M. Cancer Cells 2004, 5, 341.
since they are equal zero in the single trajectory approach. DG GB,ele is the sum of changes in the gas phase electrostatic energy and the polar solvation free energy, DE ele + DG GB . References and notes 1
DE int are not included here, since they are equal zero in the single trajectory approach. DG GB,ele is the sum of changes in the gas phase electrostatic energy and the polar solvation free energy, DE ele + DG GB. References and notes 1. Carreras, C.; Santi, D. V. Annu. Rev. Biochem. 1995, 64, 721.
  • V Tsui
  • Daj Case
Tsui V, Case DAJ. Am Chem Soc. 2000; 122:2489.
  • T E Cheatham
Cheatham, T. E., III Acc. Chem. Res. 2000, 33, 889.
  • Y Duan
  • C Wu
  • S Chowdhury
  • M C Lee
  • G Xiong
  • W Zhang
  • R Yang
  • P Cieplak
  • R Luo
  • T Lee
Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T. J. Comput. Chem. 2003, 24, 1999.
  • L Liu
  • D V Santi
Liu, L.; Santi, D. V. Biochemistry 1993, 32, 9263.
  • C I Bayly
  • P Cieplak
  • W D Cornell
  • P A Kollman
Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. J. Phys. Chem. 1993, 97, 10269.
  • J P Ryckaert
  • G Ciccotti
  • Hjc Berendsen
Ryckaert JP, Ciccotti G, Berendsen HJC. J Comput Phys. 1977; 23:327.
  • S Haider
  • G N Parkinson
  • S Neidle
Haider, S.; Parkinson, G. N.; Neidle, S. Biophys. J. 2008, 95, 296.
  • T Meyer
  • C Ferrer-Costa
  • A Perez
  • M Rueda
  • A Bidon-Chanal
  • F J Luque
  • C A Laughton
  • M Orozco
Meyer, T.; Ferrer-Costa, C.; Perez, A.; Rueda, M.; Bidon-Chanal, A.; Luque, F. J.; Laughton, C. A.; Orozco, M. J. Chem. Theory Comput. 2006, 2, 251.
  • P Cieplak
  • C I Bayly
  • W D Cornell
  • P A Kollman
Cieplak P, Bayly CI, Cornell WD, Kollman PA. J Comput Chem. 1995; 16:1357.
  • R M Stroud
  • Finer-Moore
Stroud, R. M.; Finer-Moore, J. S. Biochemistry 2003, 42, 239.
  • V Tsui
  • D A Case
Tsui, V.; Case, D. A. Biopolymers 2001, 56, 275.