Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites

AbstractSARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer’s cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain’s potential permeation pathway has six constrictions narrowing to about 1 Å in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity’s electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

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Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits

International Journal of Molecular Sciences ◽

10.3390/ijms22168891 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8891

Author(s):

Nishadh Rathod ◽

Jessi J. Bak ◽

Joseph O. Primeau ◽

M’Lynn E. Fisher ◽

Lennane Michel Espinoza-Fonseca ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Functional Properties ◽

Transmembrane Helices ◽

Regulatory Subunits ◽

Reading Frame ◽

Endoplasmic Reticulum Calcium ◽

Cellular Processes ◽

Functional Consequences ◽

Differential Binding ◽

Dynamics Simulations

The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the “regulins”. Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA—formed by transmembrane helices M2, M6, and M9—can result in distinct functional outcomes.

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Jaagsiekte Sheep Retrovirus Envelope Efficiently Pseudotypes Human Immunodeficiency Virus Type 1-Based Lentiviral Vectors

Journal of Virology ◽

10.1128/jvi.78.5.2642-2647.2003 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2642-2647 ◽

Cited By ~ 18

Author(s):

Shan-Lu Liu ◽

Christine L. Halbert ◽

A. Dusty Miller

Keyword(s):

Human Immunodeficiency Virus ◽

Epithelial Cells ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Lentiviral Vector ◽

Lung Epithelial Cells ◽

Jaagsiekte Sheep Retrovirus ◽

Lung Epithelial ◽

Immunodeficiency Virus

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) infects lung epithelial cells in sheep, and oncoretroviral vectors bearing JSRV Env can mediate transduction of human cells, suggesting that such vectors might be useful for lung-directed gene therapy. Here we show that JSRV Env can also efficiently pseudotype a human immunodeficiency virus type 1-based lentiviral vector, a more suitable vector for transduction of slowly dividing lung epithelial cells. We created several chimeric Env proteins that, unlike the parental Env, do not transform rodent fibroblasts but are still capable of pseudotyping lentiviral and oncoretroviral vectors.

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Inducible GBP5 Mediates the Antiviral Response via Interferon-Related Pathways during Influenza A Virus Infection

Journal of Innate Immunity ◽

10.1159/000460294 ◽

2017 ◽

Vol 9 (4) ◽

pp. 419-435 ◽

Cited By ~ 17

Author(s):

Jian Feng ◽

Zhongying Cao ◽

Li Wang ◽

Yushun Wan ◽

Nanfang Peng ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Lung Epithelial Cells ◽

Cellular Processes ◽

Lung Epithelial ◽

Factor Α ◽

Oxide Synthase ◽

High Degree ◽

Proinflammatory Factors

Guanylate binding protein (GBP) 5 belongs to the GBP family, which is involved in important cellular processes, including signal transduction, translation, vesicle trafficking, and exocytosis. Structurally, GBPs display a high degree of homology and share highly conserved GTP-binding or hydrolysis domains. GBP5 was reported to be a critical cellular factor in inflammasome assembly. However, little is known about its role in the host antiviral innate immune response. In this study, we found that GBP5 expression was significantly elevated in influenza patients and influenza A virus-infected A549 human lung epithelial cells. The overexpression of GBP5 inhibited virus replication by enhancing the expression of virus-induced interferon (IFN) and IFN-related effectors. Knockdown of GBP5 had the opposite effect. Moreover, GBP5 enhanced endogenous IFN expression by interacting with the NF-κB-essential modulator complex and stimulating NF-κB signaling. Additionally, the expression of proinflammatory factors, such as IL-6, IL-8, tumor necrosis factor-α, cyclooxygenase-2, and inducible nitric oxide synthase, was also activated by GBP5. Taken together, our results reveal that GBP5 inhibited virus replication through the activation of IFN signaling and proinflammatory factors.

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Pseudomonas aeruginosa Pyocyanin Is Critical for Lung Infection in Mice

Infection and Immunity ◽

10.1128/iai.72.7.4275-4278.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 4275-4278 ◽

Cited By ~ 211

Author(s):

Gee W. Lau ◽

Huimin Ran ◽

Fansheng Kong ◽

Daniel J. Hassett ◽

Dimitri Mavrodi

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Respiratory Tract ◽

Tissue Damage ◽

Lung Infection ◽

Lung Epithelial Cells ◽

Cellular Processes ◽

Redox Active ◽

Lung Epithelial

ABSTRACT Pseudomonas aeruginosa secretes copious amounts of the redox-active phenazine, pyocyanin (PCN), during cystic fibrosis lung infection. PCN has been shown to interfere with a variety of cellular processes in cultured lung epithelial cells. Here, by using two respiratory tract models of infection, we demonstrate that PCN mediates tissue damage and necrosis during lung infection.

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Towards A Molecular Understanding of The Cannabinoid Related Orphan Receptor GPR18: A Focus on Its Constitutive Activity

International Journal of Molecular Sciences ◽

10.3390/ijms20092300 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2300 ◽

Cited By ~ 3

Author(s):

Noori Sotudeh ◽

Paula Morales ◽

Dow P. Hurst ◽

Diane L. Lynch ◽

Patricia H. Reggio

Keyword(s):

G Protein ◽

Lipid Bilayers ◽

Synthetic Cannabinoids ◽

Salt Bridge ◽

Orphan Receptor ◽

Constitutive Activity ◽

Transmembrane Helices ◽

Charged Amino Acids ◽

Dynamics Simulations ◽

Oppositely Charged

The orphan G-protein coupled receptor (GPCR), GPR18, has been recently proposed as a potential member of the cannabinoid family as it recognizes several endogenous, phytogenic, and synthetic cannabinoids. Potential therapeutic applications for GPR18 include intraocular pressure, metabolic disorders, and cancer. GPR18 has been reported to have high constitutive activity, i.e., activation/signaling occurs in the absence of an agonist. This activity can be reduced significantly by the A3.39N mutation. At the intracellular (IC) ends of (transmembrane helices) TMH3 and TMH6 in GPCRs, typically, a pair of oppositely charged amino acids form a salt bridge called the “ionic lock”. Breaking of this salt bridge creates an IC opening for coupling with G protein. The GPR18 “ionic lock” residues (R3.50/S6.33) can form only a hydrogen bond. In this paper, we test the hypothesis that the high constitutive activity of GPR18 is due to the weakness of its “ionic lock” and that the A3.39N mutation strengthens this lock. To this end, we report molecular dynamics simulations of wild-type (WT) GPR18 and the A3.39N mutant in fully hydrated (POPC) phophatidylcholine lipid bilayers. Results suggest that in the A3.39N mutant, TMH6 rotates and brings R3.50 and S6.33 closer together, thus strengthening the GPR18 “ionic lock”.

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Ribonucleoprotein staining of centrioles and kinetochores in newt lung cell spindles.

The Journal of Cell Biology ◽

10.1083/jcb.80.1.1 ◽

1979 ◽

Vol 80 (1) ◽

pp. 1-9 ◽

Cited By ~ 49

Author(s):

C L Rieder

Keyword(s):

Mitotic Spindle ◽

Lung Epithelial Cells ◽

Staining Reaction ◽

Voltage Electron ◽

High Voltage Electron Microscope ◽

Lung Epithelial ◽

Pericentriolar Material ◽

Rnase Digestion ◽

Spindle Microtubules ◽

Positively Charged

The distribution of ribonucleoprotein (RNP) within the mitotic spindle of newt lung epithelial cells was studied with the high voltage electron microscope (HVEM) using Bernhard's uranyl-EDTA-lead staining of thick sections in conjunction with the ribonuclease digestion of fixed cells. The results indicate that aside from ribosomes, the major RNP-containing components of the spindle are the kinetochores and centrioles, both of which stain electron-opaque after EDTA treatment. In both cases, the electron-opaque material associated with these microtubule organizing centers (MTOC's) can be removed by RNAse digestion and cold perchloric acid (PCA) extraction under conditions which leave the spindle microtubules (Mts) centrioles, and kinetochores intact. The staining reaction is not abolished by cold PCA extraction alone or by substituting other positively charged proteins (i.e., cytochrome c or lysozyme) for RNAse. The RNP component of the kinetochore is closely associated with the bases of the kinetochore microtubules. The RNP component of the centriole can be seen to surround the microtubules of the triplet blades. No evidence was found to indicate the presence of RNP in the pericentriolar material. The possible function of both kinetochore and centriolar RNP is discussed.

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The VES KM: a pathway for protein folding in vivo

Pure and Applied Chemistry ◽

10.1515/pac-2019-0301 ◽

2020 ◽

Vol 92 (1) ◽

pp. 179-191

Author(s):

Leonor Cruzeiro

Keyword(s):

Amino Acids ◽

Protein Folding ◽

Protein Structures ◽

Kinetic Mechanism ◽

Thermal Bath ◽

Charged Amino Acids ◽

Nascent Chain ◽

Dynamics Simulations ◽

Positively Charged

AbstractWhile according to the thermodynamic hypothesis, protein folding reproducibility is ensured by the assumption that the native state corresponds to the minimum of the free energy in normal cellular conditions, here, the VES kinetic mechanism for folding in vivo is described according to which the nascent chain of all proteins is helical and the first and structure defining step in the folding pathway is the bending of that initial helix around a particular amino acid site. Molecular dynamics simulations are presented which indicate both the viability of this mechanism for folding and its limitations in the presence of a Markovian thermal bath. An analysis of a set of protein structures formed only of helices and loops suggests that bending sites are correlated with regions bounded, on the N-side, by positively charged amino acids like Lysine and Histidine and on the C-side by negatively charged amino acids like Aspartic acid.

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Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415908112 ◽

2014 ◽

Vol 112 (2) ◽

pp. E119-E126 ◽

Cited By ~ 37

Author(s):

Nabanita Das ◽

Jian Dai ◽

Ivan Hung ◽

Malini Rajagopalan ◽

Huan-Xiang Zhou ◽

...

Keyword(s):

Molecular Dynamics ◽

Mycobacterium Tuberculosis ◽

Lipid Bilayers ◽

Transmembrane Domain ◽

Magic Angle Spinning ◽

Magic Angle ◽

Transmembrane Helices ◽

Oriented Sample ◽

Angle Spinning ◽

Dynamics Simulations

The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid–crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA’s partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines.

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