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Results for “molecular dynamic simulation assay”

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PubMed2003

Development of a new family of conformationally restricted peptides as potent nucleators of beta-turns. Design, synthesis, structure, and biological evaluation of a beta-lactam peptide analogue of melanostatin.

Novel enantiopure (i)-(beta-lactam)-(Gly)-(i+3) peptide models, defined by the presence of a central alpha-alkyl-alpha-amino-beta-lactam ring placed as the (i+1) residue, have been synthesized in a totally stereocontrolled way by alpha-alkylation of suitable N-[bis(trimethylsilyl)methyl]-beta-lactams. The structural properties of these beta-lactam pseudopeptides have been studied by X-ray crystallography, Molecular Dynamics simulation, and NOESY-restrained NMR simulated annealing techniques, showing a strong tendency to form stable type II or type II' beta-turns either in the solid state or in highly coordinating DMSO solutions. Tetrapeptide models containing syn- or anti-alpha,beta-dialkyl-alpha-amino-beta-lactam rings have also been synthesized and their conformations analyzed, revealing that alpha-alkyl substitution is essential for beta-turn stabilization. A beta-lactam analogue of melanostatin (PLG amide) has also been prepared, characterized as a type-II beta-turn in DMSO-d6 solution, and tested by competitive binding assay as a dopaminergic D2 modulator in rat neuron cultured cells, displaying moderate agonist activity in the micromolar concentration range. On the basis of these results, a novel peptidomimetic design concept, based on the separation of constraint and recognition elements, is proposed.

Animals↗

PubMed2004

Structure-based discovery of nonpeptidic small organic compounds to block the T cell response to myelin basic protein.

We have utilized a computational structure-based approach to identify nonpeptidic small organic compounds that bind to a human leukocyte antigen (HLA) DR1301 molecule (HLA-DR1301 or DR1301) and block the presentation of myelin basic protein peptide 152-165 (MBP 152-165) to T cells. A three-dimensional (3D) structure of DR1301 was derived by homology modeling followed by extensive molecular dynamics simulation for structural refinement. Computational structure-based database searching was performed to identify nonpeptidic small-molecule candidates from the National Cancer Institute (NCI) database containing over 150 000 compounds that can effectively interact with the peptide-binding groove of the HLA molecule. By in vitro testing of 106 candidate small molecules, two lead compounds were confirmed to specifically block IL-2 secretion by DR1301-restricted T cells in a dose-dependent and reversible manner. The specificity of blocking DR1301-restricted MBP presentation was further validated in a binding assay using an analogue of the most potent lead compound. Computational docking was performed to predict the three-dimensional binding model of these confirmed small molecule blockers to the DR1301 molecule and to gain structural insight into their interactions. Our results suggest that computational structure-based searching is an effective approach to discover nonpeptidic small organic compounds to block the interaction between DR1301 and T cells. The nonpeptidic small organic compounds identified in this study are useful pharmacological tools to study the interactions between HLA molecules and T cells and a starting point for the development of a novel therapeutic strategy for the treatment of multiple sclerosis (MS) or other immune-related disorders.

Animals↗

PubMed2004

Ni(II).Arg-Gly-His-DNA interactions: investigation into the basis for minor-groove binding and recognition.

A study of the minor-groove recognition of A/T-rich DNA sites by Ni(II).L-Arg-Gly-His and Ni(II).D-Arg-Gly-His was carried out with a fluorescence-based binding assay, one- and two-dimensional (1D and 2D) NMR methodologies, and molecular simulations. Fluorescence displacement titrations revealed that Ni(II).L-Arg-Gly-His binds to A/T-rich sequences better than the D-Arg diastereomer, while NMR investigations revealed that both metallopeptides bind to the minor groove of an AATT core region as evidenced by an intermolecular nuclear Overhauser effect (NOE) between each metallopeptide His imidazole C4 proton and the C2 proton of adenine. Results from molecular dynamics simulations of these systems were consistent with the experimental data and indicated that the His imidazole N-H, the N-terminal peptide amine, and Arg side chains of each metallopeptide are major determinants of minor-groove recognition by functioning as H-bond donors to the O2 of thymine residues or N3 of adenine residues.

Aniline Compounds↗

PubMed2005

Modulating the DNA affinity of Elk-1 with computationally selected mutations.

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

Amino Acid Sequence↗

PubMed2006

Recent advances in molecular modeling and medicinal chemistry aspects of phospho-glycoprotein.

Phospho-glycoprotein (P-gp) is an efflux transporter expressed in many organs (ex: kidney, lung, liver and spleen) and in hormone producing or responsive tissues (ex: adrenal cortex, testis and placenta). It is involved in many important physiological functions. Among them the major one is extrusion of xenobiotics in order to detoxify the cells. This property of P-gp is associated with multidrug resistance (MDR) for many pathological conditions. While the experimental determination of three-dimensional structure is not yet successful, the transmembrane (TM) 5, 6, 11 and 12 are sensitive to mutations and contain substrate binding sites. Designing of potential and selective inhibitors of P-gp is still hampered by a lack of information upon the three dimensional structure of P-gp. The design of P-gp inhibitors was traditionally driven by quantitative structure activity relationship studies, which is complicated by factors such as different types of assays, multiple drug binding sites and diverse chemical structures. Clearly a conclusive and predictive SAR does not seem to be practical, despite progress in the last few years towards more specific SAR suggesting well defined structural features responsible for activity. Advances made recently in solving the crystal structure of prokaryotic ATP binding cassette proteins (ABC) transporters, Ec-MsbA, Vc-MsbA and BtuCD yielded suitable templates for construction of homology models of P-gp. Few molecular dynamics (MD) simulations aimed at elucidating the functional dynamics of ABC transporters have provided useful insights to their mechanism and structure. The present review aims at the general overview of importance, expression, structure, organization and drug binding sites of P-gp. This review also highlights recent developments in the homology modeling, molecular dynamics simulations of P-gp and progress in QSAR, pharmacophore modeling of P-gp modulators.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Synthesis, cytotoxic activities and proposed mode of binding of a series of bis([(9-oxo-9,10-dihydroacridine-4-carbonyl)amino]alkyl) alkylamines.

A series of bis([(9-oxo-9,10-dihydroacridine-4-carbonyl)amino]alkyl) alkylamines have been prepared and their antiproliferative properties have been tested against HT-29 cell lines. Compounds 6b and 6d showed an interesting cytotoxic profile and were subjected to further cytotoxic evaluation, DNA binding properties and molecular modelling studies. The evaluation of the cytotoxic activity of compounds 6b and 6d against pairs of cisplatin-sensitive and -resistant ovarian tumour cells shows that both compounds may be endowed with interesting antitumour properties because they are able to circumvent cisplatin resistance in A2780cisR, CH1cisR and Pam 212-ras tumour cells. On the other hand, DNA binding data indicate that compounds 6b and 6d are able to intercalate stronger than acridine within the double helix. Both compounds displace ethidium bromide with an efficiency ten times higher than acridine from several linear double-stranded DNAs and induce 43 degrees unwinding in supercoiled pBR322 DNA while acridine unwinds pBR322 DNA by only 24 degrees. Altogether these data indicate that the significant conformational changes induced by compounds 6b and 6d in the double helix are due to a bis-intercalative DNA binding mode. We propose that binding to DNA through bisintercalation might be at least in part responsible for the remarkable cytotoxic properties of these acridine derivatives. The complex of 6b with d(GCGCGC)(2) in the four possible orientations that the ligand can adopt when binding to the DNA hexamer have been modelled and subjected to molecular dynamics simulations with the aim of evaluating the binding preferences of this bisintercalating agent into the DNA molecule. The predictions suggest that 6b binds to d(GCGCGC)(2) with a parallel orientation of the chromophores relative to each other and with a preference for binding through the minor groove of the hexamer. The possible relevance of these findings to the process of bisintercalation and the antitumour profile of these compounds is discussed in this paper.

Acridines↗

PubMed1995

Conformational preferences of the calliFMRFamides and their free-acid analogues.

A molecular dynamics study was undertaken to determine the conformational basis for the differing activities of the insect neuropeptide hormones calliFMRFamide 3 (SPSQDFMRF-NH2). calliFMRFamide 5 (APGQDFMRF-NH2) and their corresponding free-acid analogues (SPSQDFM-RF-OH and APGQDFMRF-OH) in two insect bioassays. A simulated annealing protocol was used to determine the range of conformers available to the linear peptides. Analysis of the conformers obtained indicated that all the peptides exhibited distinct secondary structure preferences. These, when correlated with their biological activities, enabled the formulation of putative conformation-activity relationships for the peptides.

Amino Acid Sequence↗

PubMed1994

Stabilizing and destabilizing effects of placing beta-branched amino acids in protein alpha-helices.

In order to gain greater insight into the effects of beta-branched amino acids on protein alpha-helices, hydrophobic amino acids with varying degrees of beta-branching, including the fully beta-substituted L-2-amino-3,3-dimethylbutanoic acid (ADBA), were incorporated into the protein T4 lysozyme. The unnatural and natural amino acids were substituted at two solvent-exposed alpha-helical sites, Ser 44 and Asn 68, in the protein using the technique of unnatural amino acid mutagenesis. The stabilities of the mutant proteins were determined by using a heat of inactivation assay and from their circular dichroism thermal denaturation curves. Surprisingly, while substitution of the amino acid with the greatest degree of beta-branching, ADBA, destabilizes the protein by 2.5 +/- 0.1 degrees C (0.69 +/- 0.03 kcal/mol) relative to Ala at site 44, the same substitution stabilizes the protein by 1.0 +/- 0.1 degree C (0.27 +/- 0.03 kcal/mol) at site 68. The difference observed at these two positions illustrates the extent to which the local context can mediate the impact of a particular mutation. Molecular dynamics simulations were carried out in parallel to model the structures of the mutant proteins and to examine the energetic consequences of incorporating ADBA. Together, these results suggest that the conformationally restricted beta-branched amino acids are destabilizing, in part, because the beta-branched methyl groups can cause distortions in the local helix backbone. In addition, it is proposed that in some contexts the conformational rigidity of beta-branched amino acids may be stabilizing because it lowers the entropic cost of forming favorable side-chain van der Waals interactions.

Acetonitriles↗

PubMed2002

Structural and immunological characterisation of heteroclitic peptide analogues corresponding to the 600-612 region of the HIV envelope gp41 glycoprotein.

The conformational and immunological properties of different analogues corresponding to the 600-612 disulfide loop of the human immunodeficiency virus (HIV) gp41 glycoprotein envelope were studied. Fourteen analogues were designed and synthesised; namely, a series of seven analogues in which the disulfide bond was replaced by a lactam bridge and a series of seven analogues in which one residue of each analogue at a time, was replaced by its corresponding homologised alpha-amino acid (beta(3)-amino acid). In the case of the lactam analogues, the influence of the two possible CO-NH and NH-CO orientations of the lactam bridge as well as the size of the lactam ring was explored. The analogues were tested in ELISA with monoclonal antibodies raised against the 600-612 cyclic parent peptide as well as with sera from HIV-1 infected patients. A structural analysis of the parent and analogue peptides was carried out in dimethyl sulfoxide (DMSO-d(6)) using two-dimensional NMR techniques and molecular dynamics simulations. Comparison of the own conformation of the cyclic analogues with their either strong or weak reactivity with the antibodies reveals structural features that may be correlated with the antibody reactivity. Thus, a close structural similarity, particularly a characteristic orientation of the side-chains of residues Lys606, Leu607 and Ile608 in the loop, was found in certain beta(3)-analogues that were better recognised than the parent peptide by anti-peptide mouse monoclonal antibodies and patients' antibodies.

Amino Acid Sequence↗

PubMed2004

Solution structure and stability against digestion of rproBnIb, a recombinant 2S albumin from rapeseed: relationship to its allergenic properties.

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.

2S Albumins, Plant↗

PubMed2006

Structure-selective recognition by voltammetry: enantiomeric determination of amines using azophenolic crowns in aprotic solvent.

The enantiomeric recognition of amines by voltammetry using electroactive macrocyclic molecules, nitroazophenolic crown ethers, is reported. The oxidation potential of the nitroazophenol moiety in nitroazophenols with 18-crown-6 sensitively depends on the structure of alkyl amines. Based on this phenomenon, enantiomeric amines and even the quantitative assay of the R/S ratio in enantiomeric mixtures can be selectively recognized by using 18-crown-6 azophenol (3-H) with chiral centers. In the case of phenylglycinol, the association constants (K) of 3-H for the R and S forms have an R/S value of 3.5. The peak potential of the R form in square-wave voltammograms reproducibly differs from that of the S form by 32 mV, within which the peak potential linearly varies with the enantiomeric ratio. Free energy perturbation and molecular dynamics simulation provide deeper understanding of the enantiomeric recognition in this system. The theoretical analysis indicates that the free energy difference between diastereomeric complexes agrees well with the experimental results, and the pi-pi or charge-charge interaction plays a key role in enantiomeric recognition.

Amines↗

PubMed2001

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation.

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.

Amino Acid Substitution↗

PubMed1996

Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: leucine 66 to proline and leucine 91 to proline.

A class of temperature-sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 degrees C and no activity at 43 degrees C has been selected. These mutants, designated "tight" ts mutants, differ from most other T4 lysozyme mutants that are active at 43 degrees C, but only manifest their ts lesion by a reduced halo size around phage plaques after exposure of the growth plates to chloroform vapors. For example, in the series of T4 lysozyme mutants at position 157, the original randomly selected mutant, T1571, is the least stable of the series, yet, apart from the halo assay and subsequent in vitro protein stability measurements, this mutant is indistinguishable from wild type (WT) even at 43 degrees C. Two mutants were identified: L91P and L66P. Both insert proline residues into alpha-helical regions of the WT protein structure. The stabilities (delta delta G) as determined by urea denaturation are 8.2 kcal/mol for L91P and 7.1 kcal/mol for L66P. CD spectra indicate that no major conformational changes have occurred in the mutant structures. The structures of the mutants were modeled with a 40-ps molecular dynamics simulation using explicit solvent. For L91P, the reduction of stability appears to be due to an unsatisfied hydrogen bond in the alpha-helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain alpha-helix, at least two unsatisfied hydrogen bonds, and a newly formed solvent-filled pocket that protrudes into the hydrophobic core, possibly reducing the stabilizing contribution of a partially buried intrachain salt bridge.

Circular Dichroism↗

PubMed2002

Critical role of helix 12 of the vitamin D(3) receptor for the partial agonism of carboxylic ester antagonists.

The carboxy-terminal alpha-helix of a nuclear receptor ligand-binding domain (LBD), helix 12, contains a critical, ligand-modulated interface for the interaction with coactivator proteins. In this study, using the example of the vitamin D receptor (VDR) and the partial antagonist ZK159222, the role of helix 12 (residues 417-427) for both antagonistic and agonistic receptor actions was investigated. Amino acid residue G423 was demonstrated to be critical for partial agonism of ZK159222, but not for the activity of the natural VDR agonist, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). The amount of partial agonism of ZK159222 increased when helix 12 was truncated by the last four amino acid residues (Delta424-27) and augmented even more, when in addition helix 12 of VDR's dimerization partner, retinoid X receptor (RXR), was truncated. In contrast, the low agonism of a structural derivative of ZK159222, ZK168281, was not affected comparably, whereas other close structural relatives of ZK159222 even demonstrated the same agonistic activity as that of 1alpha,25(OH)(2)D(3). The amount of agonism of ZK159222 and ZK168281 at different variations of helix 12 correlated well with VDR's ability to complex with coactivator proteins and inversely correlated with the strength of the compound's antagonistic action on 1alpha,25(OH)(2)D(3) signalling. Molecular dynamics simulations of the LBD complexed with the two antagonists could explain their different action by demonstrating a more drastic displacement of helix 12 through ZK168281 than through ZK159222. Moreover, the modelling could indicate a kink of helix 12 at amino acid residue G423, which provides the last four amino acid residues of helix 12 with a modulatory role for the partial agonism of some VDR antagonists, such as ZK159222. In conclusion, partial agonism of a VDR antagonist is lower the more it disturbs helix 12 in taking the optimal position for coactivator interaction.

Amino Acid Substitution↗

PubMed2026

Mechanism of Action of Hedyotis diffusa Extract in a Rat Model of Acute Lung Injury Based on Transcriptomic Analysis.

OBJECTIVE: This study established a rat model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) to evaluate pathological damage, collagen deposition, inflammatory cytokine levels, and key gene/protein expression following Hedyotis diffusa water extract (HDWE) intervention. Combined with ultra-high-performance liquid chromatography-quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), transcriptomic analysis, and molecular simulation, this study identified the bioactive components of HDWE, evaluated their potential interactions with ALI-related targets, and explored the multi-omics-based protective mechanisms of HDWE. METHODS: Thirty-six Sprague-Dawley (SD) rats were randomly divided into six groups: Control group, ALI group, DXMS group, HDWE-L group (100 mg/kg), HDWE-M group (200 mg/kg), and HDWE-H group (300 mg/kg). Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to evaluate lung pathological changes and collagen deposition. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum tumor necrosis factor-α TNF-α interleukin-1β IL-1β, erleukin-6 (IL-6), and interleukin-10 (IL-10) levels. Transcriptomic analysis identified differentially expressed genes (DEGs), followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), receiver operating characteristic (ROC), and immune infiltration analyses. Quantitative real-time polymerase chain reaction (qRT-PCR) detected the mRNA expression levels of SPHK1, RELA, and NFKBIA. Immunohistochemistry evaluated the expression of eight hub targets, including endothelin-1 (EDN1), sphingosine kinase 1 (SPHK1), intercellular adhesion molecule 1 (ICAM1), interleukin-17 (IL-17), prostaglandin-endoperoxide synthase 2 (PTGS2/COX-2), NF-κB p65 (encoded by RELA), WT1-associated protein (WTAP), and myeloperoxidase (MPO). UHPLC-Q-Orbitrap HRMS characterized HDWE constituents. Molecular docking analysis was performed between 22 compounds and eight hub targets, followed by 100 ns molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) binding free energy calculations for five core targets. Compared with the control group, the ALI group showed increased levels of TNF-α (86%), IL-1β (107%), and IL-6 (66%), accompanied by a 43% reduction in IL-10 and a 300% increase in lung collagen deposition. All HDWE doses alleviated inflammatory responses, with medium-dose HDWE showing the most pronounced effects. Specifically, medium-dose HDWE increased IL-10 levels by 52% and reduced IL-6, TNF-α, and IL-1β levels by 18%, 22%, and 11%, respectively. Transcriptomic analysis identified 2512 DEGs between the control group and ALI groups, 832 exclusive DEGs between the ALI group and HDWE-M groups, and 876 overlapping DEGs enriched in TNF, IL-17, and NF-κB signaling pathways. The eight-hub-gene diagnostic model achieved an area under the curve (AUC) of 0.969. RELA, SPHK1, and four other hub genes showed positive correlations with Th1, Th17, and neutrophil infiltration. In the ALI group, SPHK1, RELA, and NFKBIA mRNA expression levels were 1.30-, 0.96-, and 0.71-fold of those in the control group, respectively. Compared with the ALI group, high-dose HDWE treatment and low-dose HDWE treatment reduced SPHK1 expression to 0.62- and 0.57-fold, respectively, and increased NFKBIA expression to 1.68- and 1.58-fold, respectively. High-dose HDWE treatment reduced RELA expression to 0.43-fold. The expression levels of inflammation-related proteins were increased in the ALI group and were reduced after HDWE treatment. Twenty-two HDWE components were identified, 16 of which met the docking criteria. Asperulosidic acid exhibited favorable predicted binding affinities with all eight targets, with calculated binding free energies of -14.74, -14.92, -17.58, -23.04, and -16.10 kcal/mol for MPO, IL-17, NF-κB p65, PTGS2/COX-2, and SPHK1, respectively. CONCLUSIONS: This study provides systematic in vivo pharmacodynamic and in silico component-target evidence regarding the protective effects of HDWE against LPS-induced ALI. HDWE treatment increased NFKBIA expression and reduced SPHK1, RELA, and multiple inflammatory protein levels, suggesting that HDWE may regulate the IL-17/NF-κB-associated inflammatory network, although direct causal relationships require further validation. Asperulosidic acid may represent a key bioactive component with broad target-binding potential. This study was limited by the use of an LPS-induced rat ALI model without gene knockout or target inhibitor validation; therefore, further functional experiments are required to confirm the proposed regulatory mechanisms.

Hedyotis diffusa↗

PubMed2003

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures.

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.

Binding Sites↗

PubMed1995

Structure of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frame shift hotspot of Salmonella typhimurium hisD3052 determined by 1H NMR and restrained molecular dynamics.

Structural refinement from solution 1H NMR data was performed on the 5'-d[ATCGC(PdG)-CGGCATG]-3'.5'-d[CATGCCGCGAT]-3' duplex, in which the adducted oligodeoxynucleotide containing the exocyclic lesion 1,N2-propano-2'-deoxyguanosine (PdG) was annealed with the complementary strand which contained a CpG deletion. The resulting duplex required PdG and one adjacent cytosine to be unpaired. A total of 352 distances were utilized to restrain molecular dynamics calculations, of which 264 were NOE-derived. These distances were calculated using complete relaxation matrix methods from hybrid matrices, which were comprised of the experimentally determined distances and additional distances derived from either A-form or B-form DNA. A simulated annealing protocol combined with the distance restraints was able to refine a single structure with an average rms deviation of < 1.35 A. The accuracy of the refined structure was assessed using full relaxation matrix calculations, which gave good agreement with measured NOE intensities. PdG was found to be stacked into the helix below base pair C3.G18, whereas C5 was found to be unpaired and extruded toward the major groove and parallel to base pair G6.C17. This created a localized bend in the DNA helix of approximately 20-35 degrees at the junction between PdG and C5. The bending corroborated previous assays performed on this modified sequence [Moe, J. G., Reddy, G. R., Marnett, L. J., & Stone, M. P. (1994) Chem. Res. Toxicol. 7, 319-328].

Base Sequence↗

PubMed2026

Characterization of a Ku-binding motif in the C-terminal region of RAG2.

We applied an unsupervised interactome analysis with the RAG2 C-terminal region (R2CT) in v-abl pro-B cells undergoing V(D)J recombination. Mass-spectrometry analyses showed that Ku70 and Ku80 were among the top 10 hits. To further strengthen these observations, we performed Proximity Ligation Assay (PLA) and characterize the existence of a GFP-R2CT-Ku complex formation in cellulo. The interaction of several partners with Ku70/80 (Ku) through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Through sequence analysis, we identified a KBM within R2CT (R-KBM, amino acids 589-527). We confirmed by calorimetry a specific micromolar interaction between this RAG2 region and Ku70/80/DNA complex. The RAG2 motif KBM can be subdivided in two conserved parts that have no interaction individually. AlphaFold2 prediction coupled with molecular dynamic simulations indicate that the C-terminal part of the RAG2 motif interacts with Ku80 on the same site than the NHEJ factor XLF. These in silico analyses indicated that the N-terminal part of the RAG2 motif interacts with DNA adjacent to Ku with a major role of the K503 residue in agreement with disruption of the interaction observed with the K503E mutant. This study further extends the large ensemble of proteins recruited at DSBs by KBM motifs and substantiates the model of a tight coupling between DNA breakage and repair during V(D)J recombination, mediated by the Ku-RAG2 C-terminus interaction.

Ku Autoantigen↗