Elucidation by NMR solution of neurotensin in small unilamellar vesicle environment: molecular surveys for neurotensin receptor recognition

Neurotensin (NT) is a tridecapeptide, hormone in the periphery and neurotransmitter in the brain. We used high-resolution nuclear magnetic resonance (NMR) to resolve the three-dimensional structure of NT in a small unilamellar vesicle (SUV) environment. We demonstrate that if the dynamic of the association–dissociation processes of peptide to SUV binding is rapid enough, structural determination can be obtained by solution NMR experiments. Thus, according to the global dynamic of the system, SUVs seem to be an effective model to mimic biological membranes, especially since the lipid composition can be modified or sterols may be added to closely mimic the biological membranes studied.

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Gating and conduction of nano-channel forming proteins: a computational approach

Monitoring conformational changes in ion channels is essential to understand their gating mechanism. Here, we explore the structural dynamics of four outer membrane proteins with different structures and functions in the slowest nonzero modes of vibration. Normal mode analysis was performed on the modified elastic network model of channel in the membrane. According to our results, when membrane proteins were analyzed in the dominant mode, the composed pores, TolC and α-hemolysin showed large motions at the intramembrane β-barrel region while, in other porins, OmpA and OmpF, largest motions observed in the region of external flexible loops. A criterion based on equipartition theorem was used to measure the possible amplitude of vibration in channel forming proteins. The current approach complements theoretical and experimental techniques including HOLE, Molecular Dynamics (MD), and voltage clamp used to address the channel’s structure and dynamics and provides the means to conduct a theoretical simultaneous study of the structure and function of the channel.

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The affinity of DNA sequences containing R5Y5 motif and TA repeats with 10.5-bp periodicity to histone octamer in vitro

Nucleosome positioning along the genome is partially determined by the intrinsic DNA sequence preferences on histone. RRRRRYYYYY (R5Y5, R?=?Purine and Y?=?Pyrimidine) motif in nucleosome DNA, which was presented based on several theoretical models by Trifonov et al., might be a facilitating sequence pattern for nucleosome assembly. However, there is not a high conformity experimental evidence to support the concept that R5Y5 motif is a key element for the determination of nucleosome positioning. In this work, the ability of the canonical, H2A.Z- and H3.3-containing octamers to assemble nucleosome on DNA templates containing R5Y5 motif and TA repeats within 10.5-bp periodicity was investigated by using salt-dialysis method in vitro. The results showed that the10.5-bp periodical distributions of both R5Y5 motif and TA repeats along DNA templates can significantly promote canonical nucleosome assembly and may be key sequence factors for canonical nucleosome assembly. Compared with TA repeats within 10.5-bp periodicity, R5Y5 motif in DNA templates did not elevate H2A.Z- and H3.3-containing nucleosome formation efficiency in vitro. This result indicates that R5Y5 motif probably isn’t a pivotal factor to regulate nucleosome assembly on histone variants. It is speculated that the regulatory mechanism of nucleosome assembly is different between canonical and variant histone. These conclusions can provide a deeper insight on the mechanism of nucleosome positioning.

Communicated by Ramaswamy H. Sarma     

Investigation on the site-selective binding of bovine serum albumin by erlotinib hydrochloride

The purpose of this study was to investigate the site-selective binding of erlotinib hydrochloride (ET), a targeted anticancer drug, to bovine serum albumin (BSA) through 1H NMR, spectroscopic, thermodynamic, and molecular modeling methods. The fluorescence quenching of BSA by ET was a result of the formation of BSA–ET complex with high binding affinity. The site marker competition study combined with isothermal titration calorimetry experiment revealed that ET binds to site II of BSA mainly through hydrogen bond and van der Waals force. Molecular docking was further applied to define the specific binding site of ET to BSA. The conformation of BSA was changed in the presence of ET, revealed by synchronous fluorescence, circular dichroism, and three-dimensional fluorescence spectroscopy results. Further, NMR analysis of the complex revealed that the binding capacity contributed by the aromatic protons in the binding site of BSA might be greater than the aliphatic protons.

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Investigation of silent information regulator 1 (Sirt1) agonists from Traditional Chinese Medicine

Silent information regulator 1 (Sirt1), a class III nicotinamide adenine dinucleotide dependent histone deacetylases, is important in cardioprotection, neuroprotection, metabolic disease, calorie restriction, and diseases associated with aging. Traditional Chinese Medicine (TCM) compounds from TCM Database@Taiwan (http://tcm.cmu.edu.tw/) were employed for screening potent Sirt1 agonists, and molecular dynamics (MD) simulation was implemented to simulate ligand optimum docking poses and protein structure under dynamic conditions. TCM compounds such as (S)-tryptophan-betaxanthin, 5-O-feruloylquinic acid, and RosA exhibited good binding affinity across different computational methods, and their drug-like potential were validated by MD simulation. Docking poses indicate that the carboxylic group of the three candidates generated H-bonds with residues in the protein chain from Ser441 to Lys444 and formed H-bond, π–cation interactions, or hydrophobic contacts with Phe297 and key active residue, His363. During MD, stable π–cation interactions with residues Phe273 or Arg274 were formed by (S)-tryptophan-betaxanthin and RosA. All candidates were anchored to His363 by stable π- or H-bonds. Hence, we propose (S)-tryptophan-betaxanthin, 5-O-feruloylquinic acid, and RosA as potential lead compounds that can be further tested in drug development process for diseases associated with aging

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Insights into the drug resistance induced by the BaDHPS mutations: molecular dynamic simulations and MM/GBSA studies

Dihydropteroate synthase (DHPS) is essential for the folic acid biosynthetic pathway in prokaryotes; the mutation forms for DHPS are found to be relative to the urgent drug resistance problems. In our study, the Bacillus anthracis DHPS (BaDHPS) was selected for molecular dynamics and binding free energy studies to investigate the biochemistry behaviors of the wild-type and mutation form BaDHPS proteins (D184N and K220Q). It is found that the conformational change of the ligand dihydropteroate sulfathiazole binding site in mutation D184N and K220Q systems is mainly attributed from the Loop 1, Loop 2, and Loop 7 regions, and the binding free energy of these mutation systems is lower than that of the wild-type system. Additionally, some important hydrogen bonds of the mutation systems are disrupted during the simulations. But the shortening of the distance between residue Thr67 and the ligand would cause significant change of the binding pose in the K220Q system. These studies of DHPS family will be helpful for further drug resistance investigations.

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Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10?Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ～2?μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect.

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Influence of divalent magnesium ion on DNA: molecular dynamics simulation studies

A large amount of experimental evidence is available on the effect of magnesium ions on the structure and stability of DNA double helix. Less is known, however, on how these ions affect the stability and dynamics of the molecule. The static time average pictures from X-ray structures or the quantum chemical energy minimized structures lack understanding of the dynamic DNA–ion interaction. The present work addresses these questions by molecular dynamics simulation studies on two DNA duplexes and their interaction with magnesium ions. Results show typical B-DNA character with occasional excursions to deviated states. We detected expected stability of the duplexes in terms of backbone conformations and base pair parameter by the CHARMM-27 force field. Ion environment analysis shows that Mg²⁺ retains the coordination sphere throughout the simulation with a preference for major groove over minor. An extensive analysis of the influence of the Mg²⁺ ion shows no evidence of the popular predictions of groove width narrowing by dipositive metal ion. The major groove atoms show higher occupancy and residence time compared to minor groove for magnesium, where no such distinction is found for the charge neutralizing Na⁺ ions. The determining factor of Mg²⁺ ion’s choice in DNA binding site evolves as the steric hindrance faced by the bulky hexahydrated cation where wider major groove gets the preference. We have shown that in case of binding of Mg²⁺ to DNA non electrostatic contributions play a major role.

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Docking and molecular dynamics studies of peptide inhibitors of ornithine decarboxylase: a rate-limiting enzyme for the metabolism of Fusarium solani

Fusarium solani causes a wide variety of diseases in plants. Polyamine biosynthesis is responsible for the growth and pathogenicity of the fungus. The initial step of this pathway involves the decarboxylation of ornithine to putrescine, and is catalyzed by the enzyme ornithine decarboxylase (ODC). Inhibiting this process may be a promising approach for the management of fungal disease in various crops. Therefore, there is a need to develop inhibitors of ODC that have higher binding capacity than ornithine. Fifteen peptides were designed and modeled based on physicochemical properties of residues in the active site of ODC. The peptide GLIWGNGPF showed the highest dock score. It is assumed that the de novo design of peptides could be a potential approach to inhibit polyamine biosynthesis. Molecular dynamics studies make an important contribution to understanding the effect of the binding of peptides and the stability of an ODC-peptide complex system.

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A possible strategy against head and neck cancer: in silico investigation of three-in-one inhibitors

Overexpression of epidermal growth factor receptor (EGFR), Her2, and uroporphyrinogen decarboxylase (UROD) occurs in a variety of malignant tumor tissues. UROD has potential to modulate tumor response of radiotherapy for head and neck cancer, and EGFR and Her2 are common drug targets for the treatment of head and neck cancer. This study attempts to find a possible lead compound backbone from TCM Database@Taiwan (http://tcm.cmu.edu.tw/) for EGFR, Her2, and UROD proteins against head and neck cancer using computational techniques. Possible traditional Chinese medicine (TCM) lead compounds had potential binding affinities with EGFR, Her2, and UROD proteins. The candidates formed stable interactions with residues Arg803, Thr854 in EGFR, residues Thr862, Asp863 in Her2 protein, and residues Arg37, Arg41 in UROD protein, which are key residues in the binding or catalytic domain of EGFR, Her2, and UROD proteins. Thus, the TCM candidates indicated a possible molecule backbone for evolving potential inhibitors for three drug target proteins against head and neck cancer.

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Insight into TPMT∗23 mutation mis-folding using molecular dynamics simulation and protein structure analysis

Thiopurine S-methyltransferase (TPMT) is an important enzyme that metabolizes thiopurine drugs. This enzyme exhibits a large number of interindividual polymorphism. TPMT^?23 polymorphism has been reported in a few cases in the world in co-dominance with TPMT^?3A. The phenotype has been reported to affect enzyme activity in vivo and in vitro. Its underlying structural basis is not clarified yet. In our study, the wild type (WT) protein structure was analyzed and the amino acids bordering water channels in thiopurine sites were identified. Molecular dynamics of both the WT and TPMT^?23 mutation was carried out. In addition, the effects of this mutation, especially on the thiopurine site which is closed with a pincer like mechanism, were investigated. We focused on explaining how a locally occurred A167G substitution propagated through hydrogen bonds alteration to induce structural modification which affects both thiopurine and S-adenosylmethionine receptors. Finally, a genetic prediction of mutation functional consequences has been conducted confirming altered activity.

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Structural insights into the South African HIV-1 subtype C protease: impact of hinge region dynamics and flap flexibility in drug resistance

The HIV protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. Resistance of HIV protease to protease inhibitors (PIs) is problematic for the effective treatment of HIV infection. The South African HIV-1 subtype C protease (C-SA PR), which contains eight polymorphisms relative to the consensus HIV-1 subtype B protease, was expressed in Escherichia coli, purified, and crystallized. The crystal structure of the C-SA PR was resolved at 2.7?Å, which is the first crystal structure of a HIV-1 subtype C protease that predominates in Africa. Structural analyses of the C-SA PR in comparison to HIV-1 subtype B proteases indicated that polymorphisms at position 36 of the homodimeric HIV-1 protease may impact on the stability of the hinge region of the protease, and hence the dynamics of the flap region. Molecular dynamics simulations showed that the flap region of the C-SA PR displays a wider range of movements over time as compared to the subtype B proteases. Reduced stability in the hinge region resulting from the absent E35-R57 salt bridge in the C-SA PR, most likely contributes to the increased flexibility of the flaps which may be associated with reduced susceptibility to PIs.

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Molecular mechanism of HIV-1 gp120 mutations that reduce CD4 binding affinity

The interaction of the HIV-1 fusion protein gp120 with its cellular receptor CD4 represents a crucial step of the viral infection process, thus rendering gp120 a promising target for the intervention with anti-HIV drugs. Naturally occurring mutations of gp120, however, can decrease its affinity for anti-infective ligands like therapeutic antibodies or soluble CD4. To understand this phenomenon on a structural level, we performed molecular dynamics simulations of two gp120 variants (termed gp120_3-2 and gp120_2-1), which exhibit a significantly decreased binding of soluble CD4. In both variants, the exchange of a nonpolar residue byglutamate was identified as an important determinant for reduced binding. However, those glutamates are located at different sequence positions and affect different steps of the recognition process: E471 in gp120_3-2 predominantly affects the CD4-bound conformation, whereas E372 in gp120_2-1 mainly modulates the conformational sampling of free gp120. Despite these differences, there exists an interesting similarity between the two variants: both glutamates exert their function by modulating the conformation and interactions of glycine-rich motifs (G366–G367, G471–G473) resulting in an accumulation of binding incompetent gp120 conformations or a loss of intermolecular gp120–CD4 hydrogen bonds. Thus, the present data suggests that interference with the structure and dynamics of glycine-rich stretches might represent a more widespread mechanism, by which gp120 mutations reduce binding affinity. This knowledge should be helpful to predict the resistance of novel gp120 mutations or to design gp120–ligands with improved binding properties.

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Interaction of Piscidin-1 with zwitterionic versus anionic membranes: a comparative molecular dynamics study

Plasma membrane of each micro-organism has a unique set of lipid composition as a consequence of the environmental adaptation or a response to exposure to antimicrobial peptides (AMPs) as antibiotic agents. Understanding the relationship between lipid composition and action of antimicrobial peptides or considering how different lipid bilayers respond to AMPs may help us design more effective peptide drugs in the future. In this contribution, we intend to elucidate how two currently used membrane models, namely palmitoyl-oleoyl-phosphtidylglycerol (POPG) and 1-palmitoyl-oleoyl-glycero-phosphocholine (POPC), respond to antimicrobial peptide Piscidin-1 (Pis-1).The computed density profile of the peptide as it moves from the bulk solvent toward the membrane core suggests that Pis-1 penetrates into the POPG bilayer less than the POPC membrane. Furthermore, we showed that the two model membranes used in this study have different behavior in the presence of Pis-1. Hence, we suggest that membrane composition could be an important factor in determining lytic ability of peptide drugs to kill a unique bacterial species.

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Remarkable disparity in mechanical response among the extracellular domains of type I and II cadherins

Cadherins, a large family of calcium-dependent adhesion molecules, are critical for intercellular adhesion. While crystallographic structures for several cadherins show clear structural similarities, their relevant adhesive strengths vary and their mechanisms of adhesion between types I and II cadherin subfamilies are still unclear. Here, stretching of cadherins was explored experimentally by atomic force microscopy and computationally by steered molecular dynamics (SMD) simulations, where partial unfolding of the E-cadherin ectodomains was observed. The SMD simulations on strand-swapping cadherin dimers displayed similarity in binding strength, suggesting contributions of other mechanisms to explain the strength differences of cell adhesion in vivo. Systematic simulations on the unfolding of the extracellular domains of type I and II cadherins revealed diverse pathways. However, at the earliest stage, a remarkable similarity in unfolding was observed for the various type I cadherins that was distinct from that for type II cadherins. This likely correlates positively with their distinct adhesive properties, suggesting that the initial forced deformation in type I cadherins may be involved in cadherin-mediated adhesion.

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Protein flexibility and conformational states of Leishmania antigen eIF-4A: identification of a novel plausible protein adjuvant using comparative genomics and molecular modeling

Recent homology modeling studies have identified specific residues (epitope) of the Leishmania RNA helicase protein (LmeIF) that stimulates production of IL-12 cytokine. However, question remains concerning how LmeIF’s N-terminal moiety initiates adjuvant effects. Extensive molecular modeling combining the normal mode analysis (NMA) and molecular dynamics simulations, in the present study, has demonstrated that the LmeIF structure may exist in two different forms corresponding to the extended and collapsed (closed) states of the entire structure. The computational results showed that the two domains of the LmeIF structure tend to undergo large fluctuations in a concerted fashion and have strong effect on the solvent accessible surface of the epitope situated on the N-terminal structure. The conformational freedom of the C-terminal domains may explain why the entire LmeIF protein is not as active as the N-terminal moiety. Thereafter, a comparative genome analysis with subsequent homology modeling and molecular electrostatic potential (MEP) techniques allowed us to predict a novel and plausible RNA helicase (LI-helicase) from the Listeria source with adjuvant property as observed for the Leishmania eIF-4A protein. The structural folding and MEP maps revealed similar topologies of the epitope of both LmeIF and LI-helicase proteins and striking identity in the local disposition of the charged groups.

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Cooperative stabilization of Zn2+:DNA complexes through netropsin binding in the minor groove of FdU-substituted DNA

The simultaneous binding of netropsin in the minor groove and Zn²⁺ in the major groove of a DNA hairpin that includes 10 consecutive FdU nucleotides at the 3′-terminus (3′FdU) was demonstrated based upon NMR spectroscopy, circular dichroism (CD), and computational modeling studies. The resulting Zn²⁺/netropsin: 3′FdU complex had very high thermal stability with aspects of the complex intact at 85?°C, conditions that result in complete dissociation of Mg²⁺ complexes. CD and ¹⁹F NMR spectroscopy were consistent with Zn²⁺ binding in the major groove of the DNA duplex and utilizing F5 and O4 of consecutive FdU nucleotides as ligands with FdU nucleotides hemi-deprotonated in the complex. Netropsin is bound in the minor groove of the DNA duplex based upon 2D NOESY data demonstrating contacts between AH2 ¹H and netropsin ¹H resonances. The Zn²⁺/netropsin: 3′FdU complex displayed increased cytotoxicity towards PC3 prostate cancer (PCa) cells relative to the constituent components or separate complexes (e.g. Zn²⁺:3′FdU) indicating that this new structural motif may be therapeutically useful for PCa treatment.

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Molecular dynamics simulations of the thermal stability of tteRBP and ecRBP

Molecular dynamics simulations were performed for investigating the thermal stability of the extremely thermophilic Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) and the mesophilic homologous Escherichia coli ribose binding protein (ecRBP). The simulations for the two proteins were carried out under the room temperature (300?K) and the optimal activity temperature (tteRBP 375?K and ecRBP 329?K), respectively. The comparative analyses of the trajectories show that the two proteins have stable overall structures at the two temperatures; further analyses indicate that they both have strong side-chain interactions and different backbone flexibilities at the different temperatures. The tteRBP 375?K and ecRBP 329?K have stronger internal motion and higher flexibility than tteRBP 300?K and ecRBP 300?K, respectively, it is noted that the flexibility of tteRBP is much higher than that of ecRBP at the two temperatures. Therefore, tteRBP 375?K can adapt to high temperature due to its higher flexibility of backbone. Combining with the researches by Cuneo et al., it is concluded that the side-chain interactions and flexibility of backbone are both the key factors to maintain thermal stability of the two proteins.

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Han ethnicity-specific type 2 diabetic treatment from traditional Chinese medicine?

Insulin-degrading enzyme (IDE) gene is one of the type 2 diabetes mellitus susceptibility genes specific to the Han Chinese population. IDE, a zinc-metalloendopeptidase, is a potential target for controlling insulin degradation. Potential lead compounds for IDE inhibition were identified from traditional Chinese medicine (TCM) through virtual screening and evaluation of their pharmacokinetic properties of absorption, distribution, metabolism, excretion, and toxicity. Molecular dynamics (MD) simulation was performed to validate the stability of complexes from docking simulation. The top three TCM compounds, dihydrocaffeic acid, isopraeroside IV, and scopolin, formed stable H–bond interactions with key residue Asn139, and were linked to active pocket residues His108, His112, and Glu189 through zinc. Torsion angle trajectories also indicated some stable interactions for each ligand with IDE. Molecular level analysis revealed that the TCM candidates might affect IDE through competitive binding to the active site and steric hindrance. Structural feature analysis reveals that high amounts of hydroxyl groups and carboxylic moieties contribute to anchor the ligand within the complex. Hence, we suggest the top three TCM compounds as potential inhibitor leads against IDE protein to control insulin degradation for type 2 diabetes mellitus.

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