scholarly journals Molecular determination of claudin-15 organization and channel selectivity

2018 ◽  
Vol 150 (7) ◽  
pp. 949-968 ◽  
Author(s):  
Priyanka Samanta ◽  
Yitang Wang ◽  
Shadi Fuladi ◽  
Jinjing Zou ◽  
Ye Li ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Three Dimensional ◽  
Ionic Currents ◽  
Selectivity Filter ◽  
Amino Acid Residues ◽  
Pass Through ◽  
Dynamics Simulations ◽  
Flux Charge ◽  
A Charge

Tight junctions are macromolecular structures that traverse the space between adjacent cells in epithelia and endothelia. Members of the claudin family are known to determine tight junction permeability in a charge- and size-selective manner. Here, we use molecular dynamics simulations to build and refine an atomic model of claudin-15 channels and study its transport properties. Our simulations indicate that claudin-15 forms well-defined channels for ions and molecules and otherwise “seals” the paracellular space through hydrophobic interactions. Ionic currents, calculated from simulation trajectories of wild-type as well as mutant channels, reflect in vitro measurements. The simulations suggest that the selectivity filter is formed by a cage of four aspartic acid residues (D55), contributed by four claudin-15 molecules, which creates a negative electrostatic potential to favor cation flux over anion flux. Charge reversal or charge ablation mutations of D55 significantly reduce cation permeability in silico and in vitro, whereas mutations of other negatively charged pore amino acid residues have a significantly smaller impact on channel permeability and selectivity. The simulations also indicate that water and small ions can pass through the channel, but larger cations, such as tetramethylammonium, do not traverse the pore. Thus, our model provides an atomic view of claudin channels, their transport function, and a potential three-dimensional organization of its selectivity filter.

Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

2020 ◽  
Author(s):  
Sean A. Newmister ◽  
Kinshuk Raj Srivastava ◽  
Rosa V. Espinoza ◽  
Kersti Caddell Haatveit ◽  
Yogan Khatri ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Molecular Basis ◽  
Hydrophobic Interactions ◽  
Cytochromes P450 ◽  
Targeted Mutagenesis ◽  
P450 Monooxygenase ◽  
Bond Functionalization ◽  
Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.<br>

scholarly journals Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

scholarly journals Computational Study of the Interaction of a PEGylated Hyperbranched Polymer/Doxorubicin Complex with a Bilipid Membrane

Fluids ◽  
2019 ◽  
Vol 4 (1) ◽  
pp. 17 ◽  
Author(s):  
Prodromos Arsenidis ◽  
Kostas Karatasos
Keyword(s):  
Hyperbranched Polymer ◽  
Lipid Membrane ◽  
Hydrophobic Interactions ◽  
Computational Study ◽  
Translational Motion ◽  
Spatial Arrangement ◽  
Aqueous Environment ◽  
Drug Molecules ◽  
Dynamics Simulations ◽  
A Charge

Fully atomistic molecular dynamics simulations are employed to study in detail the interactions between a complex comprised by a PEGylated hyperbranched polyester (HBP) and doxorubicin molecules, with a model dipalmitoylphosphatidylglycerol membrane in an aqueous environment. The effects of the presence of the lipid membrane in the drug molecules’ spatial arrangement were examined in detail and the nature of their interaction with the latter were discussed and quantified where possible. It was found that a partial migration of the drug molecules towards the membrane’s surface takes place, driven either by hydrogen-bonding (for the protonated drugs) or by hydrophobic interactions (for the neutral drug molecules). The clustering behavior of the drug molecules appeared to be enhanced in the presence of the membrane, while the development of a charge excess close to the surface of the hyperbranched polymer and of the lipid membrane was observed. The uneven charge distribution created an effective overcharging of the HBP/drug complex and the membrane/drug surface. The translational motion of the drug molecules was found to be strongly affected by the presence of the membrane. The extent of the observed changes depended on the charge of the drug molecule. The build-up of the observed charge excesses close to the surface of the polymeric host and the membrane, together with the changes in the diffusional behavior of the drug molecules are of particular interest. Both phenomena could be important at the latest stages of the liposomal disruption and the release of the drug cargo in formulations based on relevant liposomal carriers.

MOLECULAR MODELING OF THE INTERACTIONS BETWEEN HISTONE DEACETYLASE 8 AND INHIBITORS

2012 ◽  
Vol 11 (04) ◽  
pp. 907-924 ◽  
Author(s):  
DAWEI HUANG ◽  
XIAOHUI LI ◽  
ZHILONG XIU
Keyword(s):  
Molecular Dynamics ◽  
Histone Deacetylases ◽  
Hydrophobic Interactions ◽  
Binding Free Energy ◽  
Zinc Ion ◽  
Dynamics Simulation ◽  
Amino Acid Residues ◽  
Domain Docking ◽  
Dynamics Simulations ◽  
Linker Domain

Inhibitors of histone deacetylases (HDACs) have become an attractive class of anticancer agent. To understand the interaction between HDAC8 and inhibitors, including "pan-" inhibitors that inhibit many HDACs isoforms and selective inhibitors with no linker domain, docking and molecular dynamics simulation were conducted. Docking results showed the presence of π-π interactions between "linkerless" inhibitors and the aromatic amino acid residues of HDAC8 in the active site. Binding between HDAC8 and inhibitors was also stabilized by hydrogen bond and hydrophobic interaction. In molecular dynamics simulations, the zinc ion was shown to coordinate one more atom of HDAC8-"linkerless" inhibitor complexes than HDAC8-"pan-" inhibitor complexes. Persistent hydrogen bonds also existed between Tyr306 of HDAC8 and some inhibitors. When inhibitors with large cap groups bound to the active pocket of HDAC8, Phe152 and Met274 shifted from their initial positions and the entrance of the active pocket became more open, resulting in the formation of sub-pocket. Hydrophobic interactions contributed most favorably to the binding free energy between HDAC8 and inhibitors. Lys33, Asp178, Asp267, Tyr306 and Leu308 of HDAC8 were favorable for binding with all inhibitors.

scholarly journals 9-Methylfascaplysin Is a More Potent Aβ Aggregation Inhibitor than the Marine-Derived Alkaloid, Fascaplysin, and Produces Nanomolar Neuroprotective Effects in SH-SY5Y Cells

Marine Drugs ◽  
2019 ◽  
Vol 17 (2) ◽  
pp. 121 ◽  
Author(s):  
Qingmei Sun ◽  
Fufeng Liu ◽  
Jingcheng Sang ◽  
Miaoman Lin ◽  
Jiale Ma ◽  
...  
Keyword(s):  
Amino Acid Residues ◽  
Neuroprotective Effects ◽  
Study Results ◽  
Aβ Aggregation ◽  
Aggregation Inhibitors ◽  
Amyloid Aβ ◽  
Β Amyloid ◽  
Dynamics Simulations ◽  
Aβ Oligomer

β-Amyloid (Aβ) is regarded as an important pathogenic target for Alzheimer’s disease (AD), the most prevalent neurodegenerative disease. Aβ can assemble into oligomers and fibrils, and produce neurotoxicity. Therefore, Aβ aggregation inhibitors may have anti-AD therapeutic efficacies. It was found, here, that the marine-derived alkaloid, fascaplysin, inhibits Aβ fibrillization in vitro. Moreover, the new analogue, 9-methylfascaplysin, was designed and synthesized from 5-methyltryptamine. Interestingly, 9-methylfascaplysin is a more potent inhibitor of Aβ fibril formation than fascaplysin. Incubation of 9-methylfascaplysin with Aβ directly reduced Aβ oligomer formation. Molecular dynamics simulations revealed that 9-methylfascaplysin might interact with negatively charged residues of Aβ42 with polar binding energy. Hydrogen bonds and π–π interactions between the key amino acid residues of Aβ42 and 9-methylfascaplysin were also suggested. Most importantly, compared with the typical Aβ oligomer, Aβ modified by nanomolar 9-methylfascaplysin produced less neuronal toxicity in SH-SY5Y cells. 9-Methylfascaplysin appears to be one of the most potent marine-derived compounds that produces anti-Aβ neuroprotective effects. Given previous reports that fascaplysin inhibits acetylcholinesterase and induces P-glycoprotein, the current study results suggest that fascaplysin derivatives can be developed as novel anti-AD drugs that possibly act via inhibition of Aβ aggregation along with other target mechanisms.

scholarly journals Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase

2020 ◽  
Vol 295 (31) ◽  
pp. 10781-10793 ◽  
Author(s):  
Igor V. Peshenko ◽  
Alexander M. Dizhoor
Keyword(s):  
Retinal Degeneration ◽  
Guanylyl Cyclase ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Hek293 Cells ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Retinal Guanylyl Cyclase ◽  
Functional Interface

Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase–activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

2020 ◽  
Author(s):  
Sean A. Newmister ◽  
Kinshuk Raj Srivastava ◽  
Rosa V. Espinoza ◽  
Kersti Caddell Haatveit ◽  
Yogan Khatri ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Molecular Basis ◽  
Hydrophobic Interactions ◽  
Cytochromes P450 ◽  
Targeted Mutagenesis ◽  
P450 Monooxygenase ◽  
Bond Functionalization ◽  
Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.<br>

Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

2006 ◽  
Vol 387 (5) ◽  
pp. 515-523 ◽  
Author(s):  
Shivakumara Bheemanaik ◽  
Janusz M. Bujnicki ◽  
Valakunja Nagaraja ◽  
Desirazu N. Rao
Keyword(s):  
Dna Methyltransferase ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Cofactor Binding ◽  
Protein Fold Recognition ◽  
Fluorescence Measurements ◽  
Complete Disruption

AbstractKpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5′-GGTACC-3′ and transfers the methyl group fromS-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation andin vitrorestriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.

scholarly journals Discrimination of Naturally-Occurring 2-Arylbenzofurans as Cyclooxygenase-2 Inhibitors: Insights into the Binding Mode and Enzymatic Inhibitory Activity

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 176 ◽  
Author(s):  
Ericsson Coy-Barrera
Keyword(s):  
Three Dimensional ◽  
3D Qsar ◽  
Binding Mode ◽  
Binding Energies ◽  
Wide Range ◽  
Ic50 Values ◽  
Structure Relationship ◽  
Cox 2 ◽  
Dynamics Simulations

2-arylbenzofuran-containing compounds are chemical entities that can be naturally produced by several organisms. A wide-range of activities is described for several compounds of this kind and they are, therefore, valuable moieties for a lead finding from nature. Although there are in-vitro data about the activity of 2-arylbenzofuran-related compounds against cyclooxygenase (COX) enzymes, the molecular level of these COX-inhibiting constituents had not been deeply explored. Thus, 58 2-arylbenzofurans were initially screened through molecular docking within the active site of nine COX-2 crystal structures. The resulting docking scores were statistically analyzed and good reproducibility and convergence were found to discriminate the best-docked compounds. Discriminated compounds exhibited the best performance in molecular dynamics simulations as well as the most-favorable binding energies and the lowest in-vitro IC50 values for COX-2 inhibition. A three-dimensional quantitative activity-structure relationship (3D-QSAR) was also demonstrated, which showed some crucial structural requirements for enhanced enzyme inhibition. Therefore, four hits are proposed as lead structures for the development of COX-2 inhibitors based on 2-arylbenzofurans in further studies.

scholarly journals A Study of the Interaction of a New Benzimidazole Schiff Base with Synthetic and Simulated Membrane Models of Bacterial and Mammalian Membranes

Membranes ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 449
Author(s):  
Alberto Aragón-Muriel ◽  
Yamil Liscano ◽  
David Morales-Morales ◽  
Dorian Polo-Cerón ◽  
Jose Oñate-Garzón
Keyword(s):  
Molecular Dynamics ◽  
Schiff Base ◽  
Hydrophobic Interactions ◽  
Biologically Active ◽  
Membrane Model ◽  
Scanning Calorimetry ◽  
Complex Dynamic Systems ◽  
Membrane Models ◽  
Dynamics Simulations

Biological membranes are complex dynamic systems composed of a great variety of carbohydrates, lipids, and proteins, which together play a pivotal role in the protection of organisms and through which the interchange of different substances is regulated in the cell. Given the complexity of membranes, models mimicking them provide a convenient way to study and better understand their mechanisms of action and their interactions with biologically active compounds. Thus, in the present study, a new Schiff base (Bz-Im) derivative from 2-(m-aminophenyl)benzimidazole and 2,4-dihydroxybenzaldehyde was synthesized and characterized by spectroscopic and spectrometric techniques. Interaction studies of (Bz-Im) with two synthetic membrane models prepared with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and DMPC/1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) 3:1 mixture, imitating eukaryotic and prokaryotic membranes, respectively, were performed by applying differential scanning calorimetry (DSC). Molecular dynamics simulations were also developed to better understand their interactions. In vitro and in silico assays provided approaches to understand the effect of Bz-Im on these lipid systems. The DSC results showed that, at low compound concentrations, the effects were similar in both membrane models. By increasing the concentration of Bz-Im, the DMPC/DMPG membrane exhibited greater fluidity as a result of the interaction with Bz-Im. On the other hand, molecular dynamics studies carried out on the erythrocyte membrane model using the phospholipids POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), SM (N-(15Z-tetracosenoyl)-sphing-4-enine-1-phosphocholine), and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) revealed that after 30 ns of interaction, both hydrophobic interactions and hydrogen bonds were responsible for the affinity of Bz-Im for PE and SM. The interactions of the imine with POPG (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoglycerol) in the E. coli membrane model were mainly based on hydrophobic interactions.

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