L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes.

To determine if mutations introduced into phospholemman (PLM) could increase the level of PLM-Na,K-ATPase (NKA) binding, we performed scanning mutagenesis of the transmembrane domain of PLM and measured Förster resonance energy transfer (FRET) between each mutant and NKA. We observed an increased level of binding to NKA for several PLM mutants compared to that of the wild type (WT), including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca2+ transient compared to the GFP control. The Ca2+ transient amplitude was further increased by L30A PLM overexpression. The L30A mutation also delayed Ca2+ extrusion and increased the duration of cardiomyocyte contraction. This mimics aspects of the effect of cardiac glycosides, which are known to increase contractility through inhibition of NKA. No significant differences between WT and L30A PLM-expressing myocytes were observed after treatment with isoproterenol, suggesting that the superinhibitory effects of L30A are reversible with ß-adrenergic stimulation. We also observed a decrease in the extent of PLM tetramerization with L30A compared to WT using FRET, suggesting that L30 is an important residue for mediating PLM-PLM binding. Molecular dynamics simulations revealed that the potential energy of the L30A tetramer is greater than that of the WT, and that the transmembrane α helix is distorted by the mutation. The results identify PLM residue L30 as an important determinant of PLM tetramerization and of functional inhibition of NKA by PLM.

Recent discoveries of influenza A drug target sites to combat virus replication.

Sequence variations in the binding sites of influenza A proteins are known to limit the effectiveness of current antiviral drugs. Clinically, this leads to increased rates of virus transmission and pathogenicity. Potential influenza A inhibitors are continually being discovered as a result of high-throughput cell based screening studies, whereas the application of computational tools to aid drug discovery has further increased the number of predicted inhibitors reported. This review brings together the aspects that relate to the identification of influenza A drug target sites and the findings from recent antiviral drug discovery strategies.

Evaluation of a novel virtual screening strategy using receptor decoy binding sites.

Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated.

How the amyloid-ß peptide and membranes affect each other: an extensive simulation study.

The etiology of Alzheimer's disease is thought to be linked to interactions between amyloid-ß (Aß) and neural cell membranes, causing membrane disruption and increased ion conductance. The effects of Aß on lipid behavior have been characterized experimentally, but structural and causal details are lacking. We used atomistic molecular dynamics simulations totaling over 6 µs in simulation time to investigate the behavior of Aß(42) in zwitterionic and anionic lipid bilayers. We simulated transmembrane ß-sheets (monomer and tetramer) resulting from a global optimization study and a helical structure obtained from an NMR study. In all simulations Aß(42) remained embedded in the bilayer. It was found that the surface charge and the lipid tail type are determinants for transmembrane stability of Aß(42) with zwitterionic surfaces and unsaturated lipids promoting stability. From the considered structures, the ß-sheet tetramer is most stable as a result of interpeptide interactions. We performed an in-depth analysis of the translocation of water in the Aß(42)-bilayer systems. We observed that this process is generally fast (within a few nanoseconds) yet generally slower than in the peptide-free bilayers. It is mainly governed by the lipid type, simulation temperature and Aß(42) conformation. The rate limiting step is the permeation through the hydrophobic core, where interactions between Aß(42) and permeating H(2)O molecules slow the translocation process. The ß-sheet tetramer allows more water molecules to pass through the bilayer compared to monomeric Aß, allowing us to conclude that the experimentally observed permeabilization of membranes must be due to membrane-bound Aß oligomers, and not monomers.

G-quadruplex formation of FXYD1 pre-mRNA indicates the possibility of regulating expression of its protein product.

G-quadruplexes are higher-order nucleic acid structures formed of square-planar arrangements of four guanine bases held together by Hoogsteen-type hydrogen bonds. Stacks of guanine tetrads are stabilised by intercalating potassium ions. FXYD1 encodes for phospholemman, a regulatory subunit of the cardiac Na(+)/K(+)-ATPase. Computational sequence analysis of FXYD1 pre-mRNA predicted the formation of stable intramolecular G-quadruplexes in human and orthologue sequences. Multiple sequence alignment indicated that G-rich sequences are conserved in evolution suggesting a potential role of G-quadruplexes in FXYD1 gene expression. The existence of a non-functional alternative splicing product indicated that the G-quadruplex formation may control alternative splicing. Quadruplex formation of human and bovine oligonucleotides was confirmed in vitro by native polyacrylamide gel electrophoresis and intrinsic fluorescence emission spectroscopy. Taking together the evolutionary conservation of G-quadruplex forming sequences with the confirmation of G-quadruplex formation in vitro by two FXYD1 homologues the results point to a potential role of these structures in regulating the expression of FXYD1 and thus regulate indirectly the activity of the cardiac Na(+)/K(+)-ATPase.

A cellulose-based bioassay for the colorimetric detection of pathogen DNA.

Cellulose-paper-based colorimetric bioassays may be used at the point of sampling without sophisticated equipment. This study reports the development of a colorimetric bioassay based on cellulose that can detect pathogen DNA. The detection was based on covalently attached single-stranded DNA probes and visual analysis. A cellulose surface functionalized with tosyl groups was prepared by the N,N-dimethylacetamide-lithium chloride method. Tosylation of cellulose was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and elemental analysis. Sulfhydryl-modified oligonucleotide probes complementary to a segment of the DNA sequence IS6110 of Mycobacterium tuberculosis were covalently immobilized on the tosylated cellulose. On hybridization of biotin-labelled DNA oligonucleotides with these probes, a colorimetric signal was obtained with streptavidin-conjugated horseradish peroxidase catalysing the oxidation of tetramethylbenzamidine by H2O2. The colour intensity was significantly reduced when the bioassay was subjected to DNA oligonucleotide of randomized base composition. Initial experiments have shown a sensitivity of 0.1 µM. A high probe immobilization efficiency (more than 90 %) was observed with a detection limit of 0.1 µM, corresponding to an absolute amount of 10 pmol. The detection of M. tuberculosis DNA was demonstrated using this technique coupled with PCR for biotinylation of the DNA. This work shows the potential use of tosylated cellulose as the basis for point-of-sampling bioassays.

Structure and dynamics of the kinase IKK-ß--A key regulator of the NF-kappa B transcription factor.

The inhibitor κB kinase-ß (IKK-ß) phosphorylates the NF-κB inhibitor protein IκB leading to the translocation of the transcription factor NF-κB to the nucleus. The transcription factor NF-κB and consequently IKK-ß are central to signal transduction pathways of mammalian cells. The purpose of this research was to develop a 3D structural model of the IKK-ß kinase domain with its ATP cofactor and investigate its dynamics and ligand binding potential. Through a combination of comparative modelling and simulated heating/annealing molecular dynamics (SAMD) simulation in explicit water the model accuracy could be substantially improved compared to comparative modelling on its own as shown by model validation measures. The structure revealed the details of ATP/Mg(2+) binding indicating hydrophobic interactions with the adenine base and a significant contribution of Mg(2+) as a bridge between ATP phosphate groups and negatively charged side chains. The molecular dynamics trajectories of the ATP-bound and free enzyme showed two conformations in each case, which contributed to the majority of the trajectory. The ATP-free enzyme revealed a novel binding site distant from the ATP binding site that was not encountered in the ATP bound enzyme. Based on the overall structural flexibility, it is suggested that a truncated version of the kinase domain from Ala14 to Leu265 should be subjected to crystallisation trials. The 3D structure of this enzyme will enable rational design of new ligands and analysis of protein-protein interactions. Furthermore, our results may provide a new impetus for wet-lab based structural investigation focussing on a truncated kinase domain.

Integrating molecular modelling methods to advance influenza A virus drug discovery.

Since the discovery of the anti-influenza drugs oseltamivir and zanamivir using computer-aided drug design methods, there have been significant applications of molecular modelling methodologies applied to influenza A virus drug discovery, such as molecular dynamics (MD) simulation, molecular docking, and virtual screening (VS). In this review, we provide a brief general introduction to molecular modelling in the context of drug discovery and then focus on the advances and impact of integrating these methods with specific reference to potential influenza A antiviral drug targets.

Prediction of ligands to universally conserved binding sites of the influenza a virus nuclear export protein.

The nuclear export protein (NEP) of the influenza A virus exports viral ribonucleoproteins to the host cell cytoplasm following nuclear transcription. In this work conservation analysis of 3000 protein sequences and molecular modelling of full-length NEP identified ligand binding sites overlapping with high sequence conservation. Two binding hot spots were identified close to the first nuclear export signal and several hot spots overlapped with highly conserved amino acids such as Arg42, Asp43, Lys39, Ile80, Gln101 and Val109. Virtual screening with ~43,000 compounds against a binding site showed affinities of up to -8.95 kcal/mol, while ~1700 approved drugs showed affinities of up to -8.31 kcal/mol. A drug-like compounds predicted was ZINC01564229 that could be used as probe to investigate NEP function or as a new drug lead. The approved drugs Nandrolone phenylpropionate and Estropipate were predicted to bind with high affinity and may be investigated for repurposing as anti-influenza drugs. IMPORTANCE: The influenza A virus causes respiratory illness in humans and farm animals annually across the world. Antigenic shifts and drifts in the surface proteins lead to genome diversity and unpredictable pandemics and epidemics. The high evolution rate of the RNA genome can also limit the effectiveness of antivirals and is the cause of emerging resistance. From a human health perspective, it is important that compounds identified as potential influenza replication inhibitors remain effective long-term. This work presents results which are based on computational predictions that reveal interactions between available compounds and regions of the influenza A nuclear export protein which display high conservation. Due to a low probability of highly conserved regions undergoing genomic changes, these compounds may serve as ideal leads for new antivirals.

Transmembrane protein models based on high-throughput molecular dynamics simulations with experimental constraints.

Elucidating the structure of transmembrane proteins domains with high-resolution methods is a difficult and sometimes impossible task. Here, we explain the method of combining a limited amount of experimental data with automated high-throughput molecular dynamics (MD) simulations of alpha-helical transmembrane bundles in an explicit lipid bilayer/water environment. The procedure uses a systematic conformational search of the helix rotation with experimentally constrained MDs simulations. The experimentally determined helix tilt and rotational angle of a labeled residue with site-specific infrared dichroism allows us to select a unique high-resolution model from a number of possible energy minima encountered in the systematic conformational search.

Genomic evidence for genes encoding leucine-rich repeat receptors linked to resistance against the eukaryotic extra- and intracellular Brassica napus pathogens Leptosphaeria maculans and Plasmodiophora brassicae.

Genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens. However, evidence for a role of genes coding for proteins with LRR domains in resistance against extracellular (apoplastic) fungal pathogens is limited. Here, the distribution of genes coding for proteins with eLRR domains but lacking kinase domains was determined for the Brassica napus genome. Predictions of signal peptide and transmembrane regions divided these genes into 184 coding for receptor-like proteins (RLPs) and 121 coding for secreted proteins (SPs). Together with previously annotated NLRs, a total of 720 LRR genes were found. Leptosphaeria maculans-induced expression during a compatible interaction with cultivar Topas differed between RLP, SP and NLR gene families; NLR genes were induced relatively late, during the necrotrophic phase of pathogen colonization. Seven RLP, one SP and two NLR genes were found in Rlm1 and Rlm3/Rlm4/Rlm7/Rlm9 loci for resistance against L. maculans on chromosome A07 of B. napus. One NLR gene at the Rlm9 locus was positively selected, as was the RLP gene on chromosome A10 with LepR3 and Rlm2 alleles conferring resistance against L. maculans races with corresponding effectors AvrLm1 and AvrLm2, respectively. Known loci for resistance against L. maculans (extracellular hemi-biotrophic fungus), Sclerotinia sclerotiorum (necrotrophic fungus) and Plasmodiophora brassicae (intracellular, obligate biotrophic protist) were examined for presence of RLPs, SPs and NLRs in these regions. Whereas loci for resistance against P. brassicae were enriched for NLRs, no such signature was observed for the other pathogens. These findings demonstrate involvement of (i) NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.

The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics.

ErbB-2 is a member of the family of epidermal growth factor receptors, which shows an oncogenic mutation in the rat gene neu, Val664Glu in the transmembrane domain that causes permanent dimerisation and subsequently leads to uncontrollable cell division and tumour formation. We have obtained the alpha-helical structure of the mutant transmembrane domain dimer experimentally with site-specific infrared dichroism (SSID) based on six transmembrane peptides with 13C18O carbonyl group-labelled residues. The derived orientational data indicate a local helix tilt ranging from 28(+/-6) degrees to 22(+/-4) degrees. Altogether using orientational constraints from SSID and experimental alpha-helical constraints while performing a systematic conformational search including molecular dynamics simulation in a lipid bilayer, we have obtained a unique experimentally defined atomic structure. The resulting structure consists of a right handed alpha-helical bundle with the residues Ile659, Val663, Leu667, Ile671, Val674 and Leu679 in the dimerisation interface. The right-handed bundle is in contrast to the left-handed structures obtained in previous modelling efforts. In order to facilitate tight helical packing, the spacious Glu664 residues do not interact directly but with water molecules that enter the bilayer.

Evolutionary conservation of influenza A PB2 sequences reveals potential target sites for small molecule inhibitors.

The influenza A basic polymerase protein 2 (PB2) functions as part of a heterotrimer to replicate the viral RNA genome. To investigate novel PB2 antiviral target sites, this work identified evolutionary conserved regions across the PB2 protein sequence amongst all sub-types and hosts, as well as ligand binding hot spots which overlap with highly conserved areas. Fifteen binding sites were predicted in different PB2 domains; some of which reside in areas of unknown function. Virtual screening of ~50,000 drug-like compounds showed binding affinities of up to -10.3kcal/mol. The highest affinity molecules were found to interact with conserved residues including Gln138, Gly222, Ile529, Asn540 and Thr530. A library containing 1738 FDA approved drugs was screened additionally and revealed Paliperidone as a top hit with a binding affinity of -10kcal/mol. Predicted ligands are ideal leads for new antivirals as they were targeted to evolutionary conserved binding sites.

Secondary structure, orientation, and oligomerization of phospholemman, a cardiac transmembrane protein.

Human phospholemman (PLM) is a 72-residue protein, which is expressed at high density in the cardiac plasma membrane and in various other tissues. It forms ion channels selective for K+, Cl-, and taurine in lipid bilayers and colocalizes with the Na+/K+-ATPase and the Na+/Ca2+-exchanger, which may suggest a role in the regulation of cell volume. Here we present the first structural data based on synthetic peptides representing the transmembrane domain of PLM. Perfluoro-octaneoate-PAGE of reconstituted proteoliposomes containing PLM reveals a tetrameric homo-oligomerization. Infrared spectroscopy of proteoliposomes shows that the PLM peptide is completely alpha-helical, even beyond the hydrophobic core residues. Hydrogen/deuterium exchange experiments reveal that a core of 20-22 residues is not accessible to water, thus embedded in the lipid membrane. The maximum helix tilt is 17 degrees +/- 2 degrees obtained by attenuated total reflection infrared spectroscopy. Thus, our data support the idea of ion channel formation by the PLM transmembrane domain.

Systematic molecular dynamics searching in a lipid bilayer: application to the glycophorin A and oncogenic ErbB-2 transmembrane domains.

Molecular dynamics (MD) simulations of proteins in a lipid bilayer environment are usually undertaken with one or a few starting structures. Here we report a search protocol for systematically exploring the possible interactions in helical bundle transmembrane proteins, a frequently occurring structural motif. The search protocol correctly identifies the experimentally known structure of the dimeric human glycophorin A transmembrane domain as the lowest energy structure among five different models without any prior assumptions, whilst an identical in vacuo search fails to identify the correct structure. The lowest energy structure from the search in a lipid bilayer has a root mean square deviation of 1.1A to the experimental structure. We have applied the same search protocol to the unknown transmembrane structure of the oncogenic mutant ErbB-2 protein, a member of the family of epidermal growth factor receptors. Resulting structures show the role of glutamic acid hydrogen bonding and close helical packing. Water molecules may also play a key role in stabilisation of the transmembrane helix association.

Conformational flexibility of the peptide hormone ghrelin in solution and lipid membrane bound: a molecular dynamics study.

Human ghrelin is a peptide hormone of 28 aminoacid residues, in which the Ser3 is modified by an octanoyl group. Ghrelin has a major role in the energy metabolism of the human body stimulating growth hormone release as well as food intake. Here we perform molecular dynamics simulations in explicit water and in a DMPC-lipid bilayer/water system in order to structurally characterize this highly flexible peptide and its lipid binding properties. We find a loop structure with residues Glu17 to Lys 20 in the bending region and a short alpha-helix from residues Pro7 to Glu13. The presence of a lipid membrane does not influence these structural features, but reduces the overall flexibility of the molecule as revealed by reduced root mean square fluctuations of the atom coordinates. The octanoyl-side chain does not insert into the lipid membrane but points into the water phase. The peptide binds to the lipid membrane with its bending region involving residues Arg15, Lys16, Glu17, and Ser18. The implications of these results for the binding pocket of the ghrelin receptor are discussed.

A structure for the trimeric MHC class II-associated invariant chain transmembrane domain.

The major histocompatibility complex (MHC)-associated invariant chain (Ii) contains a single transmembrane domain that forms trimers. Ii is involved in the assembly of the MHC and antigen presentation, and is thus central to the function of the immune system. Here, we show by attenuated total reflectance, Fourier transform infrared (ATR-FTIR) spectroscopy that the transmembrane domain is alpha-helical and we provide a structural model of the transmembrane domain obtained by a combination of site-specific infrared dichroism and molecular dynamics (MD) simulations. This work resolves the backbone structure of a transmembrane peptide by multiple (13)C=(18)O labelling at ten different residues. A second purely computational approach, based on MD simulations of Ii transmembrane homologous sequences, yields a similar structure that is consistent with our experimental results. The structure presented forms a left-handed coiled coil with an average helix tilt of 13(+/-6) degrees; the residue Gln47 implicated in trimer formation forms strong interhelical contacts, Thr50 points to the inside of the trimeric coil and forms a network of hydrogen bonds.

Lipid membranes for membrane proteins.

The molecular dynamics (MD) simulation of membrane proteins requires the setup of an accurate representation of lipid bilayers. This chapter describes the setup of a lipid bilayer system from scratch using generally available tools, starting with a definition of the lipid molecule POPE, generation of a lipid bilayer, energy minimization, MD simulation, and data analysis. The data analysis includes the calculation of area and volume per lipid, deuterium order parameters, self-diffusion constant, and the electron density profile.

Large-scale analysis of influenza A virus nucleoprotein sequence conservation reveals potential drug-target sites.

The nucleoprotein (NP) of the influenza A virus encapsidates the viral RNA and participates in the infectious life cycle of the virus. The aims of this study were to find the degree of conservation of NP among all virus subtypes and hosts and to identify conserved binding sites, which may be utilised as potential drug target sites. The analysis of conservation based on 4430 amino acid sequences identified high conservation in known functional regions as well as novel highly conserved sites. Highly variable clusters identified on the surface of NP may be associated with adaptation to different hosts and avoidance of the host immune defence. Ligand binding potential overlapping with high conservation was found in the tail-loop binding site and near the putative RNA binding region. The results provide the basis for developing antivirals that may be universally effective and have a reduced potential to induce resistance through mutations.

Consensus virtual screening approaches to predict protein ligands.

In order to exploit the advantages of receptor-based virtual screening, namely time/cost saving and specificity, it is important to rely on algorithms that predict a high number of active ligands at the top ranks of a small molecule database. Towards that goal consensus methods combining the results of several docking algorithms were developed and compared against the individual algorithms. Furthermore, a recently proposed rescoring method based on drug efficiency indices was evaluated. Among AutoDock Vina 1.0, AutoDock 4.2 and GemDock, AutoDock Vina was the best performing single method in predicting high affinity ligands from a database of known ligands and decoys. The rescoring of predicted binding energies with the water/octanol partition coefficient did not lead to an improvement averaged over ten receptor targets. Various consensus algorithms were investigated and a simple combination of AutoDock and AutoDock Vina results gave the most consistent performance that showed early enrichment of known ligands for all receptor targets investigated. In case a number of ligands is known for a specific target, every method proposed in this study should be evaluated.