Modeling of Peptides in Implicit Membrane-Mimetic Media

Abstract

Lipid bilayer plays a crucial role in folding of membrane peptides and their stabilization in the membrane-bound state. Correct treatment of the media effects is thus essential for realistic simulations of peptides in bilayers. Previously (Volynsky et al., 1999), we proposed an efficient solvation model which mimics heterogeneous membrane-water system. The model is based on combined employment of atomic solvation parameters for water and hydrocarbon, which approximate hydrated headgroups and acyl chains of lipids, respectively. In this study, the model is employed in non-restrained Monte Carlo simulations of several peptides: totally apolar 20-residue poly-L-Leu, hydrophobic peptide with polar edges, and strongly amphiphilic pep-tide. The principal goals are: to explore energy landscape of these peptides in membrane; to characterize the structures of low-energy states and their orientations with respect to the bilayer. Simulations were performed starting from different structures (unordered or helical) and orientations. It was found that the membrane environment significantly promotes an α-helical conformation for all the peptides, while their energetically favourable orientations are quite different. Thus, poly-Leu was immobilized inside the membrane, the hydrophobic peptide with polar termini adapted transbilayer orientation, whereas the amphiphilic peptide stayed on the lipid-water interface in peripherial orientation. Energy barriers between different states were characterized. The computational results were compared with the experimental structural data.     

Complexes of antiparallel telomeric G-quadruplex d(TTAGGG)4 with carboxymethyl tetracationic porphyrins

Porphyrins are a chemical class that is widely used in drug design. Cationic porphyrins may bind to DNA guanine quadruplexes. We report the parameters of the binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium) porphyrin (P1) and 5,10,15,20-tetrakis(N-etoxycarbonylmethyl-4-pyridinium) porphyrin (P2) to antiparallel telomeric G-quadruplex formed by d(TTAGGG)₄ sequence (TelQ). The binding constants (K _i ) and the number of binding sites (N _j ) were determined from absorption isotherms generated from the absorption spectra of complexes of P1 and P2 with TelQ. Compound P1 demonstrated a high affinity to TelQ (K _i = (40 ± 6) × 10⁶ M^?1, N ₁ = 1; K ₂ = (5.4 ± 0.4) × 10⁶ M^?1, N ₂ = 2). In contrast, the binding constants of P2-TelQ complexes (K ₁ = (3.1 ± 0.2) × 10⁶ M^?1, N ₁ = 1; K ₂ = (1.2 ± 0.2) × 10⁶ M^?1, N ₂ = 2) were one order of magnitude smaller than the corresponding values for P2-TelQ complexes. Measurements of the quantum yield and fluorescence lifetime of the drug’s TelQ complexes revealed two types of binding sites for P1 and P2 on the quadruplex oligonucleotide. We concluded that strong complexes can result from the interaction of the porphyrins with TTA loops whereas the weaker complexes are formed with G-quartets. The altered TelQ conformation detected by the circular dichroism spectra of P1-TelQ complexes can be explained by the disruption of the G-quartet. We conclude that peripheral carboxy groups contribute to the high affinity of P1 for the antiparallel telomeric G-quadruplex.     

Coagulation Factor Binding Orientation and Dimerization May Influence Infectivity of Adenovirus-Coagulation Factor Complexes

Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus.     

Regulatory role of membrane fluidity in gene expression and physiological functions

Plants, algae, and photosynthetic bacteria experience frequent changes in environment. The ability to survive depends on their capacity to acclimate to such changes. In particular, fluctuations in temperature affect the fluidity of cytoplasmic and thylakoid membranes. The molecular mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the understanding of the functions of the individual genes for fatty acid desaturases in cyanobacteria and plants led to the directed mutagenesis of such genes that altered the membrane fluidity of cytoplasmic and thylakoid membranes. Characterization of the photosynthetic properties of the transformed cyanobacteria and higher plants revealed that lipid unsaturation is essential for protection of the photosynthetic machinery against environmental stresses, such as strong light, salt stress, and high and low temperatures. The unsaturation of fatty acids enhances the repair of the damaged photosystem II complex under stress conditions. In this review, we summarize the knowledge on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, on genes that are involved in acclimation to new sets of environmental conditions, and on the influence of membrane properties on photosynthetic functions.     

Ca is involved in phytochrome A-dependent regulation of the succinate dehydrogenase gene sdh1-2 in Arabidopsis

The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis has been investigated. Using the phytochrome mutants of Arabidopsis, it is demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca²⁺ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene pif3 encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene sdh1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that Ca²⁺ ions are involved in the phytochrome A-mediated inhibition of succinate dehydrogenase activity in the light.     

Two-component covalent inhibitor

Inhibitors that covalently damage proteins or nucleic acids offer great potency, but are difficult to rationally design and suffer from poor specificity. Here we outline a general concept for constructing covalent inhibitors, called the two-component covalent inhibitor (TCCI). The approach takes advantage of two ligand analogs equipped with pre-reactive groups. Binding of the analogs to the adjacent sites of a target biopolymer brings the pre-reactive groups in close proximity and causes their interaction followed by covalent damage of the target. In the present study we used light-activated pre-reactive groups to inactivate a DNA polymerase. It was found that the efficiency of a traditional single-component inhibitor was greatly reduced in the presence of a non-target protein, while the TCCI was not significantly affected. Our findings suggest that TCCI approach has advantages in inactivation of biopolymers in complex multi-component systems.     

Discovery of subtype selective muscarinic receptor antagonists as alternatives to atropine using in silico pharmacophore modeling and virtual screening methods

Muscarinic acetylcholine receptors (mAChRs) have five known subtypes which are widely distributed in both the peripheral and central nervous system for regulation of a variety of cholinergic functions. Atropine is a well known muscarinic subtype non-specific antagonist that competitively inhibits acetylcholine (ACh) at postganglionic muscarinic sites. Atropine is used to treat organophosphate (OP) poisoning and resulting seizures in the warfighter because it competitively inhibits acetylcholine (ACh) at the muscarinic cholinergic receptors. ACh accumulates due to OP inhibition of acetylcholinesterase (AChE), the enzyme that hydrolyzes ACh. However, atropine produces several unwanted side-effects including dilated pupils, blurred vision, light sensitivity, and dry mouth. To overcome these side-effects, our goal was to find an alternative to atropine that emphasizes M1 (seizure prevention) antagonism but has minimum M2 (cardiac) and M3 (e.g., eye) antagonism so that an effective less toxic medical countermeasure may be developed to protect the warfighter against OP and other chemical warfare agents (CWAs). We adopted an in silico pharmacophore modeling strategy to develop features that are characteristics of known M1 subtype-selective compounds and used the model to identify several antagonists by screening an in-house (WRAIR-CIS) compound database. The generated model for the M1 selectivity was found to contain two hydrogen bond acceptors, one aliphatic hydrophobic, and one ring aromatic feature distributed in a 3D space. From an initial identification of about five hundred compounds, 173 compounds were selected through principal component and cluster analyses and in silico ADME/Toxicity evaluations. Next, these selected compounds were evaluated in a subtype-selective in vitro radioligand binding assay. Twenty eight of the compounds showed antimuscarinic activity. Nine compounds showed specificity for M1 receptors and low specificity for M3 receptors. The pK_i values of the compounds range from 4.5 to 8.5 nM in comparison to a value of 8.7 nM for atropine. 2-(diethylamino)ethyl 2,2-diphenylpropanoate (ZW62841) was found have the best desired selectivity. None of the newly found compounds were previously reported to exhibit antimuscarinic specificity. Both theoretical and experimental results are presented.