Biology subjects

Tajkhorshid, E.

Publications and source records attributed to Tajkhorshid, E..

At least 55 records · Page 3Linked to original sources

bioRxiv2020

Active Participation of Membrane Lipids in Inhibition of Multidrug Transporter P-Glycoprotein

P-glycoprotein (Pgp) is a major efflux pump in humans, overexpressed in a variety of cancers and associated with the development of multi-drug resistance. Allosteric modulation by various ligands (e.g., transport substrates, inhibitors, and ATP) has been biochemically shown to directly influence structural dynamics, and thereby, the function of Pgp. However, the molecular details of such effects, particularly with respect to the role and involvement of the surrounding lipids, are not well established. Here, we employ all-atom molecular dynamics (MD) simulations to study the conformational landscape of Pgp in the presence of a high-affinity, third-generation inhibitor, tariquidar, in comparison to the nucleotide-free (APO) and the ATP-bound states, in order to characterize the mechanical effects of the inhibitor that might be of relevance to its blocking mechanism of Pgp. Simulations in a multi-component lipid bilayer show a dynamic equilibrium between open(er) and more closed inward-facing (IF) conformations in the APO state, with binding of ATP shifting the equilibrium towards conformations more prone to ATP hydrolysis and subsequent events in the transport cycle. In the presence of the inhibitor bound to the drug-binding pocket within the transmembrane domain (TMD), Pgp samples more open IF conformations, and the nucleotide binding domains (NBDs) become highly dynamic. Interestingly, and reproduced in multiple independent simulations, the inhibitor is observed to facilitate recruitment of lipid molecules into the Pgp lumen through the two proposed drug-entry portals, where the lipid head groups from the cytoplasmic leaflet penetrate into and, in some cases, translocate inside the TMD, while the lipid tails remain extended into the bulk lipid environment. These "wedge" lipids likely enhance the inhibitor-induced conformational restriction of the TMD leading to the differential modulation of coupling pathways observed with the NBDs downstream. We suggest a novel inhibitory mechanism for tariquidar, and potentially for related third-generation Pgp inhibitors, where lipids are seen to enhance the inhibitory role in the catalytic cycle of membrane transporters.

biophysics↗

bioRxiv2020

Binding Mode of SARS-CoV2 Fusion Peptide to Human Cellular Membrane

Infection of human cells by the SARS-CoV2 relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics (MD) simulations taking advantage of the Highly Mobile Membrane Mimetic (HMMM) model, to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level, and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration where the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane binding modes (binding modes 1-3) where binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the "fusion active core residues", in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection with potential implications in designing novel inhibitors. SignificanceA key step in cellular infection by the SARS-CoV2 virus is its attachment to and penetration into the plasma membrane of human cells. These processes hinge upon the membrane interaction of the viral fusion peptide, a segment exposed by the spike protein upon its conformational changes after encountering the host cell. In this study, using molecular dynamics simulations, we describe how the fusion peptide from the SARS-CoV2 virus binds human cellular membranes and characterize, at an atomic level, lipid-protein interactions important for the stability of its membrane-bound state.

biophysics↗

bioRxiv2020

How to build a water-splitting machine: structural insights into photosystem II assembly

Biogenesis of photosystem II (PSII), natures water splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate with 2.94 [A] resolution. It contains three assembly factors (Psb27, Psb28, Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace bicarbonate with glutamate as a ligand of the non-heme iron, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water splitting reaction. One Sentence HighlightThe high-resolution Cryo-EM structure of the photosystem II assembly intermediate PSII-I reveals how natures water splitting catalyst is assembled, protected and prepared for photoactivation by help of the three assembly factors Psb27, Psb28 and Psb34.

plant biology↗

bioRxiv2020

A statistical physics based approach to capture spurious solutions in AI-enhanced molecular dynamics

Artificial intelligence (AI)-based approaches have had indubitable impact across the sciences through the ability to extract relevant information from raw data. Recently AI has also seen use for enhancing the efficiency of molecular simulations, wherein AI derived slow modes are used to accelerate the simulation in targeted ways. However, while typical fields where AI is used are characterized by a plethora of data, molecular simulations per-construction suffer from limited sampling and thus limited data. As such the use of AI in molecular simulations can suffer from a dangerous situation where the AI-optimization could get stuck in spurious regimes, leading to incorrect characterization of the reaction coordinate (RC) for the problem at hand. When such an incorrect RC is then used to perform additional simulations, one could start to deviate progressively from the ground truth. To deal with this problem of spurious AI-solutions, here we report a novel and automated algorithm using ideas from statistical mechanics. It is based on the notion that a more reliable AI-solution will be one that maximizes the time-scale separation between slow and fast processes. To learn this time-scale separation even from limited data, we use a maximum caliber-based framework. We show the applicability of this automatic protocol for 3 classic benchmark problems, namely the conformational dynamics of a model peptide, ligand-unbinding from a protein, and folding/unfolding energy landscape of the C-terminal domain of protein G. We believe our work will lead to increased and robust use of trustworthy AI in molecular simulations of complex systems.

biophysics↗

bioRxiv2020

Glutamate transporters contain a conserved chloride channel with two hydrophobic gates

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity1. Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2-5. Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport6-8. However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

biophysics↗

bioRxiv2020

Anti-inflammatory dopamine- and serotonin-based endocannabinoid epoxides reciprocally regulate cannabinoid receptors and the TRPV1 channel

The endocannabinoid (eCB) system is a promising target to mitigate pain as the eCBs are endogenous ligands of the pain-mediating receptors--cannabinoid receptors 1 and 2 (CB1 and CB2) and TRPV1. Here we report on a novel class of lipids formed by the epoxidation of N-arachidonoyl-dopamine (NADA) and N-arachidonoyl serotonin (NA5HT) by cytochrome P450 (CYP) epoxygenases. These epoxides (epoNADA and epoNA5HT) are dual-functional rheostat modulators (varying strength of agonism or antagonism) of the eCB-TRPV1 axis. In fact, epoNADA is a 6-fold stronger agonist of TRPV1 than NADA while epoNA5HT is a 30-fold stronger antagonist of TRPV1 than NA5HT and displays a significantly stronger inhibition on TRPV1-mediated responses in primary afferent neurons. Moreover, epoNA5HT is a full CB1 agonist. The epoxides reduce the pro-inflammatory biomarkers IL-6, IL-1{beta}, TNF- and nitrous oxide (NO) and raise anti-inflammatory IL-10 in activated microglial cells. The epoxides are spontaneously generated by activated microglia cells and their formation is potentiated in the presence of another eCB, anandamide (AEA). We provide evidence for the direct biochemical mechanism of this potentiation using human CYP2J2, a CYP epoxygenase in the human brain, using detailed kinetics studies and molecular dynamics simulations. Taken all together, inflammation leads to an increase in the metabolism of NADA, NA5HT and other eCBs by CYP epoxygenases to form the corresponding epoxides. The epoxide metabolites are bioactive lipids that are more potent, multi-faceted endogenous molecules, capable of influencing the activity of CB1, CB2 and TRPV1 receptors. The identification of these molecules will serve as templates for the synthesis of new multi-target therapeutics for the treatment of inflammatory pain.

biochemistry↗

bioRxiv2020

Computational Recipe for Designing Antibodies against the Ebola Virus

A conceptual basis for antiviral therapy is to deliver a synthetic antibody that binds to a viral surface protein, and thus prevents the virus from deploying its cell-entry mechanism. The fast and untraceable virus mutations take lives of thousands of people before the immune system can produce the inhibitory antibody. In this paper, we devised a computational recipe to predict both the viral escape mutations and the possible inhibitory synthetic antibodies. We combined bioinformatics, structural biology, and molecular dynamics (MD) simulations to explore the most likely viral mutations and the candidate antibodies that can inhibit those escape mutations. Specifically, using the crystal structures of the Sudan and Zaire Ebola viral GPs in complex to their respective antibodies (ABs), we have performed an extensive set of MD simulations, both on the wild-type structures and on a large array of additional complexes designed and generated through combinatorial mutations. We discovered that our methods enabled the successful redesign of antibody sequences to essentially all likely glycoprotein mutations. Our findings and the computational methodology developed here for general antibody design can facilitate therapy of current and possibly next generations of viruses. Significance of the ManuscriptThis manuscript has high significance both methodologically and in potential biomedical application. In methodology, the manuscript combines molecular dynamics, Monte Carlo calculations, and bioinformatics in a novel way to simulate the evolutionary arms race between an evolving viral coat protein and a counter-evolving antibody against the virus. This simulation is shown to provide a method for designing a synthetic antibody against the newly emerging viral strains. This work is done in the context of ongoing work in other laboratories in which cells can be induced to produce synthetic antibodies and those synthetic antibodies can be edited (via, for example, CRISPR) to have an arbitrary sequence in the region that binds the viral coat protein. Putting those experimental methods together with the computational methods we present in this paper has the potential to provide a important approach to produce antibodies-on-demand against evolving viruses.

immunology↗

bioRxiv2020

Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

biochemistry↗

bioRxiv2020

The Structures of Secretory and Dimeric Immunoglobulin A

Secretory (S) Immunoglobulin (I) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a {beta}-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen binding fragments (Fabs) and preserves steric accessibility to receptor binding sites, likely influencing antigen binding and effector functions.

immunology↗

bioRxiv2019

Structural characterization of an intermediate reveals a unified mechanism for the CLC Cl-/H+ transport cycle

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize many experimental findings, there have remained gaps and inconsistencies in our understanding. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 [A] structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to a state that has improved accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Based on this new structure, together with DEER measurements, MD simulations, and functional studies, we propose a unified model of the CLC transport mechanism that reconciles existing data on all CLC-type proteins.

biophysics↗

bioRxiv2019

Non-Canonical Binding of a Small Molecule to Sortilin Alters Cellular Trafficking of ApoB and PCSK9 in Liver Derived Cells

Sortilin regulates hepatic exocytosis and endocytosis of ApoB containing lipoproteins (ApoB-Lp) and mediates the secretion of the subtilase PCSK9. To elucidate connections between these pathways, we previously identified a small molecule (cpd984) that binds to a non-canonical site on Sortilin. In hepatic cells cpd984 augments ApoB-Lp secretion, increases cellular PCSK9 levels, and reduces LDLR expression indicative of reduced secretion of PCSK9. We have shown that insulin-induced ApoB-Lp degradation occurs through Vps34-dependent autophagy. Here we show that the specific Vps34 inhibitor PIK-III enhances ApoB-100 secretion, reducing cellular levels of PCSK9 and Sortilin resulting in reduced LDLR expression, which implicates a role for autophagy in PCSK9 secretion. Results suggest that Sortilin is central to both PCSK9 and ApoB-100 secretion. Finally, we found that cpd984 in yeast blocks CPY secretion while increasing vacuolar homotypic fusion in a Vps10-dependent manner, indicating an evolutionarily conserved mechanism required for lysosomal protease trafficking.

biochemistry↗

bioRxiv2019

Mechanism of gating and partial agonist action in the glycine receptor

The glycine receptor is a pentameric, neurotransmitter-activated ion channel that transitions between closed/resting, open and desensitized states. Glycine, a full agonist, produces an open channel probability (Po) of [~]1.0 while partial agonists, such as taurine and {gamma}-amino butyric acid (GABA) yield submaximal Po values. Despite extensive studies of pentameric Cys-loop receptors, there is little knowledge of the molecular mechanisms underpinning partial agonist action and how the receptor transitions from the closed to open and to desensitized conformations. Here we use electrophysiology and molecular dynamics (MD) simulations, together with a large ensemble of single particle cryo-EM reconstructions, to show how agonists populate agonist-bound yet closed channel states, thus explaining their lesser efficacy, yet also populate agonist-bound open and desensitized states. Measurements within the neurotransmitter binding pocket, as a function of bound agonist, provide a metric to correlate the extent of agonist-induced conformational changes to open channel probability across the Cys-loop receptor family.

neuroscience↗

bioRxiv2019

Aβ(1-42) tetramer and octamer structures reveal edge pores as a mechanism for membrane damage

The formation of amyloid-beta (A{beta}) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimers disease (AD). It is therefore critical to characterize A{beta} oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an A{beta} oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an A{beta}(1-42) tetramer. It comprises a {beta}-sheet core made of six {beta}-strands, connected by only two {beta}-turns. The two faces of the {beta}-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of A{beta}(1-42), we prepared a sample enriched in A{beta}(1-42) octamers, formed by two A{beta}(1-42) tetramers facing each other forming a {beta}-sandwich structure. Notably, samples enriched in A{beta}(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core {beta}-sheets edges of the A{beta}(1-42) tetramers and octamers.

biophysics↗

bioRxiv2019

CryoFold: Ab-initio structure determination from electron density maps using molecular dynamics

Cryo-EM is a powerful method for determining protein structures. But it requires computational assistance. Physics-based computations have the power to give low-free-energy structures and ensembles of populations, but have been computationally limited to only small soluble proteins. Here, we introduce CryoFold. By integrating data of varying sparsity from electron density maps of 3-5 [A] resolution with coarse-grained physical knowledge of secondary and tertiary interactions, CryoFold determines ensembles of protein structures directly from sequence. We give six examples showing its broad capabilities, over proteins ranging from 72 to 2000 residues, including membrane and multi-domain proteins, and including results from two EMDB competitions. The ensembles CryoFold predicts starting from the density data of a single known protein conformation encompass multiple low-energy conformations, all of which are experimentally validated and biologically relevant.

biophysics↗

bioRxiv2019

Overexpression of a non-muscle RBFOX2 isoform triggers cardiac conduction defects in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by a CTG trinucleotide repeat expansion in the 3' untranslated region of DMPK gene. Heart dysfunctions occur in nearly 80% of DM1 patients and are the second leading cause of DM1-related deaths. Despite these figures, the mechanisms underlying cardiac-based DM1 phenotypes are unknown. Herein, we report that upregulation of a non-muscle splice isoform of RNA binding protein RBFOX2 in DM1 heart tissue--due to altered splicing factor and microRNA activities--induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX2 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac-conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from both DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX2-driven splicing defects in the voltage-gated sodium and potassium channels, which can alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX2 isoform in DM1 cardiac pathogenesis.

genetics↗

bioRxiv2019

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca2+-activated Cl- Channel ANO1 (TMEM16A)

ANO1 (TMEM16A) is a Ca2+-activated Cl- channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Here we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid-protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 {micro}M. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2. Three of these sites captured 85% of all ANO1 - PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane-cytosol interface found three regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same three sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid sidechains and phosphate/hydroxyl groups on PI(4,5)P2. Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl-ions. We propose a model consisting of a network of three PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.\n\nSignificance statementMembrane proteins dwell in a sea of phospholipids that not only structurally stabilize the proteins by providing a hydrophobic environment for their transmembrane segments, but also dynamically regulate protein function. While many cation channels are known to be regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), relatively little is known about anion channel regulation by phosphoinositides. Using a combination of patch clamp electrophysiology and atomistic molecular dynamics simulations, we have identified several PI(4,5)P2 binding sites in ANO1 (TMEM16A), a Cl- channel that performs myriad physiological functions from epithelial fluid secretion to regulation of electrical excitability. These binding sites form a band at the cytosolic interface of the membrane that we propose constitute a network to dynamically regulate this highly allosteric protein.

biophysics↗

bioRxiv2018

Selective Permeability of Carboxysome Shell Pores to Anionic Molecules

Carboxysomes are closed polyhedral cellular microcompartments that increase the efficiency of carbon fixation in autotrophic bacteria. Carboxysome shells consist of small proteins that form hexameric units with semi-permeable central pores containing binding sites for anions. This feature is thought to selectively allow access to RuBisCO enzymes inside the carboxysome by [Formula] (the dominant form of CO2 in the aqueous solution at pH 7.4) but not O2, which leads to a non-productive reaction. To test this hypothesis, here we use molecular dynamics simulations to characterize the energetics and permeability of CO2, O2, and [Formula] through the central pores of two different shell proteins, namely, CsoS1A of -carboxysome and CcmK4 of {beta}-carboxysome shells. We find that the central pores are in fact selectively permeable to anions such as [Formula], as predicted by the model.

biochemistry↗

bioRxiv2018

Phosphatidic acid inhibits SNARE priming by inducing conformational changes in Sec18 protomers

Eukaryotic homeostasis relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are re-activated by Sec18/NSF driven disassembly to enable a new round of fusion. We previously found that phosphatidic acid (PA) binds Sec18 to sequester it from SNAREs. Dephosphorylation of PA dissociates Sec18 from the membrane allowing it to engage SNARE complexes. We now report that PA induces conformational changes in Sec18 protomers, while hexameric Sec18 cannot bind PA membranes. The association of Sec18 with PA was shown to be sensitive to membrane curvature, suggesting that regulation could vary on different organelles in a curvature dependent manner. Molecular dynamics showed that PA binding sites exist on the D1 and D2 domains of Sec18 and that residues needed for binding were masked in the hexameric form of the protein. Together these data indicate that PA regulates Sec18 function through altering protein architecture and stabilizing membrane-bound protomers.

biochemistry↗