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1.
Sci Rep ; 13(1): 9166, 2023 06 06.
Article in English | MEDLINE | ID: mdl-37280310

ABSTRACT

A growing body of evidence suggests that oxysterols such as 25-hydroxycholesterol (25HC) are biologically active and involved in many physiological and pathological processes. Our previous study demonstrated that 25HC induces an innate immune response during viral infections by activating the integrin-focal adhesion kinase (FAK) pathway. 25HC produced the proinflammatory response by binding directly to integrins at a novel binding site (site II) and triggering the production of proinflammatory mediators such as tumor necrosis factor-α (TNF) and interleukin-6 (IL-6). 24-(S)-hydroxycholesterol (24HC), a structural isomer of 25HC, plays a critical role in cholesterol homeostasis in the human brain and is implicated in multiple inflammatory conditions, including Alzheimer's disease. However, whether 24HC can induce a proinflammatory response like 25HC in non-neuronal cells has not been studied and remains unknown. The aim of this study was to examine whether 24HC produces such an immune response using in silico and in vitro experiments. Our results indicate that despite being a structural isomer of 25HC, 24HC binds at site II in a distinct binding mode, engages in varied residue interactions, and produces significant conformational changes in the specificity-determining loop (SDL). In addition, our surface plasmon resonance (SPR) study reveals that 24HC could directly bind to integrin αvß3, with a binding affinity three-fold lower than 25HC. Furthermore, our in vitro studies with macrophages support the involvement of FAK and NFκB signaling pathways in triggering 24HC-mediated production of TNF. Thus, we have identified 24HC as another oxysterol that binds to integrin αvß3 and promotes a proinflammatory response via the integrin-FAK-NFκB pathway.


Subject(s)
Hydroxycholesterols , Integrin alphaVbeta3 , Computer Simulation , Humans , Integrin alphaVbeta3/chemistry , Integrin alphaVbeta3/metabolism , Hydroxycholesterols/chemistry , Hydroxycholesterols/metabolism , Inflammation/metabolism , Signal Transduction , Macrophages/metabolism , Models, Molecular , Thermodynamics , Protein Conformation , Surface Plasmon Resonance , Cholesterol 24-Hydroxylase/metabolism
2.
Biophys J ; 122(5): 849-867, 2023 03 07.
Article in English | MEDLINE | ID: mdl-36721367

ABSTRACT

In mammalian cells, all-trans farnesol, a 15-carbon isoprenol, is a product of the mevalonate pathway. It is the natural substrate of alcohol dehydrogenase and a substrate for CYP2E1, two enzymes implicated in ethanol metabolism. Studies have shown that farnesol is present in the human brain and inhibits voltage-gated Ca2+ channels at much lower concentrations than ethanol. Here we show that farnesol modulates the activity of γ-aminobutyric acid type A receptors (GABAARs), some of which also mediate the sedative activity of ethanol. Electrophysiology experiments performed in HEK cells expressing human α1ß3γ2 or α6ß3γ2 GABAARs revealed that farnesol increased chloride currents through positive allosteric modulation of these receptors and showed dependence on both the alcoholic functional group of farnesol and the length of the alkyl chain for activity. In silico studies using long-timescale unbiased all-atom molecular dynamics (MD) simulations of the human α1ß3γ2 GABAA receptors revealed that farnesol modulates the channel by directly binding to the transmembrane neurosteroid-binding site, after partitioning into the surrounding membrane and reaching the receptor by lateral diffusion. Channel activation by farnesol was further characterized by several structural and dynamic variables, such as global twisting of the receptor's extracellular domain, tilting of the transmembrane M2 helices, radius, cross-sectional area, hydration status, and electrostatic potential of the channel pore. Our results expand the pharmacological activities of farnesol to yet another class of ion channels implicated in neurotransmission, thus providing a novel path for understanding and treatment of diseases involving GABAA receptor dysfunction.


Subject(s)
Neurosteroids , Receptors, GABA-A , Humans , Binding Sites , Farnesol/pharmacology , gamma-Aminobutyric Acid/pharmacology , Protein Domains , Receptors, GABA-A/metabolism
3.
Stem Cell Reports ; 17(9): 2023-2036, 2022 09 13.
Article in English | MEDLINE | ID: mdl-35931078

ABSTRACT

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I-IV, with the most N-terminal domain involving residues 77-466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants. Isogenic control iPSCs were generated using CRISPR-Cas9/PiggyBac. Fluo-4 Ca2+ imaging assessed Ca2+ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca2+ sparking and Ca2+ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca2+ amplitude and sparking in variant iPSC-CMs. Molecular dynamic simulations visualized the structural role of these variants. We provide the first functional evidence that these most proximal N-terminal localizing variants alter calcium handling similar to CPVT1. These variants are located at the N-terminal domain and the central domain interface and could destabilize the RYR2 channel promoting Ca2+ leak-triggered arrhythmias.


Subject(s)
Induced Pluripotent Stem Cells , Ryanodine Receptor Calcium Release Channel , Tachycardia, Ventricular , Arrhythmias, Cardiac/pathology , Calcium/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Isoproterenol , Mutation , Myocytes, Cardiac/metabolism , NAD , Ryanodine Receptor Calcium Release Channel/genetics , Tachycardia, Ventricular/drug therapy , Tachycardia, Ventricular/genetics , Tachycardia, Ventricular/pathology
4.
J Clin Invest ; 132(13)2022 07 01.
Article in English | MEDLINE | ID: mdl-35617047

ABSTRACT

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.


Subject(s)
Mitochondrial Diseases , Ribonucleotide Reductases , DNA Replication , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Humans , Mitochondrial Diseases/genetics , Nucleosides , Nucleotides/genetics , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleoside Diphosphate Reductase/metabolism , Ribonucleotide Reductases/genetics , Ribonucleotide Reductases/metabolism
5.
Mol Pharmacol ; 100(4): 406-427, 2021 10.
Article in English | MEDLINE | ID: mdl-34334369

ABSTRACT

The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment of asthma and other chronic pulmonary diseases. These drugs activate the ß2-adrenergic receptors (ß2-AR), a class A G protein-coupled receptor (GPCR), and differ significantly in their clinical onset and duration of actions. According to the microkinetic model, the long duration of action of salmeterol and formoterol compared with salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classic and enhanced molecular dynamics simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known to be critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other ß2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process. SIGNIFICANCE STATEMENT: The cell membrane's functional role behind the duration of action of long-acting ß2-adrenergic receptor (ß2-AR) agonists such as salmeterol has been a subject of debate for a long time. This study investigated the binding and unbinding mechanisms of the three commonly used ß2-AR agonists, salmeterol, formoterol, and salbutamol, using advanced simulation techniques. The obtained results offer unprecedented insights into the active role of membrane lipids in facilitating access and binding of the ligands, affecting the molecular recognition process and thus their pharmacology.


Subject(s)
Adrenergic beta-2 Receptor Agonists/chemistry , Adrenergic beta-2 Receptor Agonists/metabolism , Cell Membrane/metabolism , Molecular Docking Simulation/methods , Albuterol/chemistry , Albuterol/metabolism , Binding Sites/physiology , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/metabolism , Formoterol Fumarate/chemistry , Formoterol Fumarate/metabolism , Humans , Protein Binding/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Salmeterol Xinafoate/chemistry , Salmeterol Xinafoate/metabolism
6.
Drug Metab Dispos ; 48(10): 1028-1043, 2020 10.
Article in English | MEDLINE | ID: mdl-32788161

ABSTRACT

Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.


Subject(s)
Acrolein/analogs & derivatives , Cinnamomum zeylanicum/chemistry , Cytochrome P-450 CYP2A6/antagonists & inhibitors , Herb-Drug Interactions , Acrolein/chemistry , Acrolein/pharmacology , Area Under Curve , Cytochrome P-450 CYP2A6/isolation & purification , Cytochrome P-450 CYP2A6/metabolism , Cytochrome P-450 CYP2A6/ultrastructure , Drug Evaluation, Preclinical , Humans , Letrozole/pharmacokinetics , Microsomes, Liver , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Nicotine/pharmacokinetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
7.
Mol Pharm ; 17(6): 2155-2164, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32374613

ABSTRACT

Resolvins D1 and D2 (RvDs) are structural isomers and metabolites of docosahexaenoic acid, an omega-3 fatty acid, enzymatically produced in our body in response to acute inflammation or microbial invasion. Resolvins have been shown to play an essential role in the resolution of inflammation, tissue repair, and return to homeostasis and thus are actively pursued as potential therapeutics in treating inflammatory disorders and infectious diseases. However, effective in vivo delivery of RvDs continues to be a challenging task. Recent studies demonstrated that RvD1 or RvD2 loaded in cell membrane-derived nanovesicles significantly increased therapeutic efficacy in treating murine peritonitis and ischemic stroke, respectively. The mechanistic details of how the subtle structural difference between RvD1 and RvD2 alters their molecular interactions with the membrane lipids of the nanovesicles and thus affects the loading efficiency remain unknown. Here, we report the encapsulation profiles of the neutral and ionized species of both RvD1 and RvD2 determined with the cell membrane-derived nanovesicles at pH values 5.4 and 7.4, respectively. Also, we performed microsecond time-scale all-atom molecular dynamics (MD) simulations in explicit water to elucidate the molecular interactions of both neutral and ionized species of RvD1 and RvD2 with the lipid bilayer using a model membrane system, containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. We found that the differences in the position and chirality of hydroxyl groups in RvD1 and RvD2 affected their location, orientation, and conformations within the bilayer. Surprisingly, the deprotonation of their carboxyl group caused their orientation and conformation to change from a fully extended one that is oriented in parallel to the membrane plane to a J-shaped bent conformation that is oriented perpendicular to the bilayer plane. Our studies offer valuable insight into the molecular interactions of RvD1/D2 with the lipid bilayer in atomistic details and provide a mechanistic explanation for the observed differences in the encapsulation profiles of RvD1 and RvD2, which may facilitate the rational design of nanovesicle-based therapeutics for treating inflammatory diseases.


Subject(s)
Docosahexaenoic Acids/chemistry , Molecular Dynamics Simulation , Cholesterol/chemistry , Lipid Bilayers/chemistry , Nanoparticles/chemistry , Nanotechnology/methods , Phosphatidylcholines/chemistry
8.
Nat Commun ; 10(1): 1482, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30931941

ABSTRACT

Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5ß1 and αvß3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvß3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.


Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/metabolism , Hydroxycholesterols/metabolism , Immunity, Innate/immunology , Integrin alpha5beta1/immunology , Integrin alphaVbeta3/immunology , Macrophages/immunology , Animals , Focal Adhesions , Inflammation , Integrin alpha5beta1/metabolism , Integrin alphaVbeta3/metabolism , Interleukin-6/immunology , Macrophages/metabolism , Mice , Mice, Knockout , Receptors, Pattern Recognition/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/immunology
9.
Methods Mol Biol ; 1958: 297-311, 2019.
Article in English | MEDLINE | ID: mdl-30945225

ABSTRACT

A relatively recently discovered class of proteins known as transformer proteins undergo large-scale conformational conversions that change their supersecondary structure. These structural transformations lead to different configurations that perform different functions. We describe computational methods using molecular dynamics simulations that allow the determination of the specific amino acids that facilitate the conformational transformations. These investigations provide guidance on the location and type of amino acid mutations that can either enhance or inhibit the structural transitions that allow transformer proteins to perform multiple functions.


Subject(s)
Amino Acid Motifs , Computational Biology/methods , Proteins/chemistry , Amino Acid Sequence/genetics , Molecular Dynamics Simulation , Mutation/genetics , Protein Multimerization , Proteins/genetics
10.
Mol Pharmacol ; 96(5): 527-541, 2019 11.
Article in English | MEDLINE | ID: mdl-30967440

ABSTRACT

The ligand-binding sites of many G protein-coupled receptors (GPCRs) are situated around and deeply embedded within the central pocket formed by their seven transmembrane-spanning α-helical domains. Generally, these binding sites are assumed accessible to endogenous ligands from the aqueous phase. Recent advances in the structural biology of GPCRs, along with biophysical and computational studies, suggest that amphiphilic and lipophilic molecules may gain access to these receptors by first partitioning into the membrane and then reaching the binding site via lateral diffusion through the lipid bilayer. In addition, several crystal structures of class A and class B GPCRs bound to their ligands offer unprecedented details on the existence of lipid-facing allosteric binding sites outside the transmembrane helices that can only be reached via lipid pathways. The highly organized structure of the lipid bilayer may direct lipophilic or amphiphilic drugs to a specific depth within the bilayer, changing local concentration of the drug near the binding site and affecting its binding kinetics. Additionally, the constraints of the lipid bilayer, including its composition and biophysical properties, may play a critical role in "pre-organizing" ligand molecules in an optimal orientation and conformation to facilitate receptor binding. Despite its clear involvement in molecular recognition processes, the critical role of the membrane in binding ligands to lipid-exposed transmembrane binding sites remains poorly understood and warrants comprehensive investigation. Understanding the mechanistic basis of the structure-membrane interaction relationship of drugs will not only provide useful insights about receptor binding kinetics but will also enhance our ability to take advantage of the apparent membrane contributions when designing drugs that target transmembrane proteins with improved efficacy and safety. In this minireview, we summarize recent structural and computational studies on membrane contributions to binding processes, elucidating both lipid pathways of ligand access and binding mechanisms for several orthosteric and allosteric ligands of class A and class B GPCRs.


Subject(s)
Allosteric Site/physiology , Ligands , Lipid Bilayers/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Binding Sites/physiology , Humans , Lipid Bilayers/chemistry , Protein Binding/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/chemistry
11.
J Biol Chem ; 293(9): 3335-3349, 2018 03 02.
Article in English | MEDLINE | ID: mdl-29348171

ABSTRACT

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures (i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys224, 225 and Lys274, 275). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes.


Subject(s)
Ebolavirus/metabolism , Phosphatidylserines/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism , Cell Membrane/metabolism , Ebolavirus/physiology , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Domains , Protein Multimerization , Protein Structure, Quaternary , Protein Transport , Substrate Specificity , Viral Matrix Proteins/genetics
12.
Biochem Biophys Res Commun ; 493(1): 176-181, 2017 11 04.
Article in English | MEDLINE | ID: mdl-28917841

ABSTRACT

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.


Subject(s)
Graphite/chemistry , Nucleoproteins/chemistry , Nucleoproteins/ultrastructure , Viral Core Proteins/chemistry , Viral Core Proteins/ultrastructure , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/ultrastructure , Binding Sites , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Multimerization
13.
Biochim Biophys Acta Biomembr ; 1859(10): 2012-2020, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28711356

ABSTRACT

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle.


Subject(s)
Cell Membrane/metabolism , Ebolavirus/metabolism , Lipid Bilayers/metabolism , Lipids/physiology , Nucleoproteins/metabolism , Viral Core Proteins/metabolism , Anions/metabolism , Hemorrhagic Fever, Ebola/metabolism , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Phosphatidylserines/metabolism , Protein Conformation, alpha-Helical , Protein Multimerization/physiology , Virus Release/physiology
14.
RSC Adv ; 7(37): 22741-22748, 2017 Apr 26.
Article in English | MEDLINE | ID: mdl-28580138

ABSTRACT

Filovirus infections cause hemorrhagic fever in humans and non-human primates that often results in high fatality rates. The Marburg virus is a lipid-enveloped virus from the Filoviridae family and is closely related to the Ebola virus. The viral matrix layer underneath the lipid envelope is formed by the matrix protein VP40 (VP40), which is also involved in other functions during the viral life-cycle. As in the Ebola virus VP40 (eVP40), the recently determined X-ray crystal structure of the Marburg virus VP40 (mVP40) features loops containing cationic residues that form a lipid binding basic patch. However, the mVP40 basic patch is significantly flatter with a more extended surface than in eVP40, suggesting the possibility of differences in the plasma membrane interactions and phospholipid specificity between the VP40 dimers. In this paper, we report on molecular dynamics simulations that investigate the roles of various residues and lipid types in PM association as well as the conformational changes of the mVP40 dimer facilitated by membrane association. We compared the structural changes of the mVP40 dimer with the mVP40 dimer in both lipid free and membrane associated conditions. Despite the significant structural differences in the crystal structure, the Marburg VP40 dimer is found to adopt a configuration very similar to the Ebola VP40 dimer after associating with the membrane. This conformational rearrangement upon lipid binding allows Marburg VP40 to localize and stabilize at the membrane surface in a manner similar to the Ebola VP40 dimer. Consideration of the structural information in its lipid-interacting condition may be important in targeting mVP40 for novel drugs to inhibit viral budding from the plasma membrane.

15.
Phys Chem Chem Phys ; 18(41): 28409-28417, 2016 Oct 19.
Article in English | MEDLINE | ID: mdl-27757455

ABSTRACT

The Ebola virus is a lipid-enveloped virus that obtains its lipid coat from the plasma membrane of the host cell it infects during the budding process. The Ebola virus protein VP40 localizes to the inner leaflet of the plasma membrane and forms the viral matrix, which provides the major structure for the Ebola virus particles. VP40 is initially a dimer that rearranges to a hexameric structure that mediates budding. VP40 hexamers and larger filaments have been shown to be stabilized by PI(4,5)P2 in the plasma membrane inner leaflet. Reduction in the plasma membrane levels of PI(4,5)P2 significantly reduce formation of VP40 oligomers and virus-like particles. We investigated the lipid-protein interactions in VP40 hexamers at the plasma membrane. We quantified lipid-lipid self-clustering by calculating the fractional interaction matrix and found that the VP40 hexamer significantly enhances the PI(4,5)P2 clustering. The radial pair distribution functions suggest a strong interaction between PI(4,5)P2 and the VP40 hexamer. The cationic Lys side chains are found to mediate the PIP2 clustering around the protein, with cholesterol filling the space between the interacting PIP2 molecules. These computational studies support recent experimental data and provide new insights into the mechanisms by which VP40 assembles at the plasma membrane inner leaflet, alters membrane curvature, and forms new virus-like particles.


Subject(s)
Cell Membrane/chemistry , Ebolavirus , Nucleoproteins/chemistry , Phospholipids/chemistry , Viral Core Proteins/chemistry , Computer Simulation , Models, Molecular
16.
Protein Sci ; 25(9): 1648-58, 2016 09.
Article in English | MEDLINE | ID: mdl-27328459

ABSTRACT

The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C-terminal domain (CTD) from the N-terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain-domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt-bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt-bridge between D45-K326 when the CTD participates in a latch-like interaction with the NTD. The D45-K326 salt-bridge interaction is proposed to help domain-domain association, whereas the E76-K291 interaction is important for stabilizing the closed-form structure. The effects of the removal of important VP40 salt-bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP-tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation.


Subject(s)
Cell Membrane , Ebolavirus , Protein Multimerization , Viral Matrix Proteins , Amino Acid Substitution , Cell Membrane/chemistry , Cell Membrane/genetics , Cell Membrane/metabolism , Ebolavirus/chemistry , Ebolavirus/genetics , Ebolavirus/metabolism , HEK293 Cells , Humans , Mutation, Missense , Protein Domains , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism
17.
J Phys Chem B ; 119(40): 12750-9, 2015 Oct 08.
Article in English | MEDLINE | ID: mdl-26374226

ABSTRACT

The transcription antiterminator RfaH has been shown to undergo major structural rearrangements to perform multiple functions. Structural determination of the C-terminal domain (CTD) of RfaH showed that it can exist as either an α-helix bundle when interfacing with the N-terminal domain (NTD) or as a ß-barrel conformation when it is not interfacing with the NTD. In this paper, we investigate the full RfaH with both CTD and NTD using a variety of all-atom molecular dynamics (MD) simulation techniques, including targeted molecular dynamics, steered molecular dynamics, and adaptive biasing force, and calculate potentials of mean force. We also use network analysis to determine communities of amino acids that are important in transferring information about structural changes. We find that the CTD-NTD interdomain interactions constitute the main barrier in the CTD α-helix to ß-barrel structural conversion. Once the interfacial interactions are broken, the structural conversion of the CTD is relatively easy. We determined which amino acids play especially important roles in controlling the interdomain motions and also describe subtle structural changes that may be important in the functioning of RfaH.


Subject(s)
Escherichia coli Proteins/chemistry , Peptide Elongation Factors/chemistry , Trans-Activators/chemistry , Crystallography, X-Ray , Molecular Dynamics Simulation , Protein Conformation
18.
J Phys Chem B ; 118(19): 5101-8, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24758259

ABSTRACT

The C-terminal domain (CTD) of the transcription antiterminator RfaH folds to an α-helix bundle when it interacts with its N-terminal domain (NTD) but it undergoes an all-α to all-ß conformational transformation when it does not interact with the NTD. The RfaH-CTD in the all-α topology is involved in regulating transcription whereas in the all-ß topology it is involved in stimulating translation by recruiting a ribosome to an mRNA. Because the conformational transformation in RfaH-CTD gives it a different function, it is labeled as a transformer protein, a class that may eventually include many other functional proteins. The structure and function of RfaH is of interest for its own sake, as well as for the value it may serve as a model system for investigating structural transformations in general. We used replica exchange molecular dynamics simulations with implicit solvent to investigate the α-helix to ß-structure transformation of RfaH-CTD, followed by structural relaxation with detailed all atom simulations for the best replica. The importance of interfacial interactions between the two domains of RfaH is highlighted by the compromised structural integrity of the helical form of the CTD in the absence NTD. Calculations of free-energy landscape and transfer entropy elucidate the details of the RfaH-CTD transformation process.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Molecular Dynamics Simulation , Peptide Elongation Factors/chemistry , Trans-Activators/chemistry , Escherichia coli/metabolism , Kinetics , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Static Electricity , Thermodynamics
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