Divalent cation-induced conformational changes of influenza virus hemagglutinin

Abstract Divalent cations Cu2+ and Zn2+ can prevent the viral growth in mammalian cells during influenza infection, and viral titers decrease significantly on a copper surface. The underlying mechanisms include DNA damage by radicals, modulation of viral protease, M1 or neuraminidase, and morphological changes in viral particles. However, the molecular mechanisms underlying divalent cation-mediated antiviral activities are unclear. An unexpected observation of this study was that a Zn2+ ion is bound by Glu68 and His137 residues at the head regions of two neighboring trimers in the crystal structure of hemagglutinin (HA) derived from A/Thailand/CU44/2006. The binding of Zn2+ at high concentrations induced multimerization of HA and decreased its acid stability. The acid-induced conformational change of HA occurred even at neutral pH in the presence of Zn2+. The fusion of viral and host endosomal membranes requires substantial conformational changes in HA upon exposure to acidic pH. Therefore, our results suggest that binding of Zn2+ may facilitate the conformational changes of HA, analogous to that induced by acidic pH.

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Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence

eLife ◽

10.7554/elife.06792 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 15

Author(s):

Kevin G Hicks ◽

Scott P Delbecq ◽

Enea Sancho-Vaello ◽

Marie-Pierre Blanc ◽

Katja K Dove ◽

...

Keyword(s):

Disulfide Bond ◽

Conformational Changes ◽

Divalent Cation ◽

Histidine Kinase ◽

Divalent Cations ◽

Conformational Flexibility ◽

Structural Flexibility ◽

Acidic Ph ◽

Ph Sensing ◽

Cation Sensing

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQW104C-A128C is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQW104C-A128C Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

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Influx of calcium, strontium, and barium in presynaptic nerve endings.

The Journal of General Physiology ◽

10.1085/jgp.79.6.1065 ◽

1982 ◽

Vol 79 (6) ◽

pp. 1065-1087 ◽

Cited By ~ 75

Author(s):

D A Nachshen ◽

M P Blaustein

Keyword(s):

Divalent Cation ◽

Binding Sites ◽

Divalent Cations ◽

Ion Selectivity ◽

Permeability Ratio ◽

Low Concentrations ◽

Slow Channel ◽

Channel Selectivity ◽

High Concentrations ◽

Two Phases

Depolarization-induced (potassium-stimulated) influx of 45Ca, 85Sr, and 133Ba was measured in synaptosomes prepared from rat brain. There are two phases of divalent cation entry, "fast" and "slow;" each phase is mediated by channels with distinctive characteristics. The fast channels inactivate (within 1 s) and are blocked by low concentrations (less than 1 micro M) of La. The slow channels do not inactivate (within 10 s), and are blocked by high concentrations (greater than 50 micro M) of La. Divalent cation influx through both channels saturates with increasing concentrations of permeant divalent cation; in addition, each permeant divalent cation species competitively blocks the influx of other permeant species. These results are consistent with the presence of "binding sites" for divalent cations in the fast and slow channels. The Ca:Sr:Ba permeability ratio, determined by measuring the influx of all three species in triple-label experiments, was 6:3:2 for the fast channel and 6:3:1 for the slow channel. A simple model for ion selectivity, based on the presence of a binding site in the channel, could account well for slow and, to some extent, for fast, channel selectivity data.

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Evolution of Differences in Transport Function in Slc11a Family Members

Journal of Biological Chemistry ◽

10.1074/jbc.m707057200 ◽

2007 ◽

Vol 282 (49) ◽

pp. 35646-35656 ◽

Cited By ~ 30

Author(s):

Michala Eichner Techau ◽

Javier Valdez-Taubas ◽

Jean-François Popoff ◽

Richard Francis ◽

Matthew Seaman ◽

...

Keyword(s):

Divalent Cation ◽

Mammalian Cells ◽

Transmembrane Domain ◽

Divalent Cations ◽

Transport Function ◽

Cation Uptake ◽

Cation Homeostasis ◽

N Terminus ◽

Resistance To Infection ◽

Divalent Cation Transporter

Slc11a1 (formerly Nramp1) is a proton/divalent cation transporter that regulates cation homeostasis in macrophages. Slc11a2 mediates divalent cation uptake via the gut and delivery into cells. The mode of action of the two transporters remains controversial. Heterologous expression in frog oocytes shows Slc11a2 is a symporter, whereas Slc11a1 is an antiporter fluxing divalent cations against the proton gradient. This explains why Slc11a2, but not Slc11a1, can complement EGTA sensitivity in smf1Δ/smf2Δ/smf3Δ yeast. However, some studies of transport in mammalian cells suggest Slc11a1 is a symporter. We now demonstrate that Slc11a1, but not Slc11a2, complements a divalent cation stress phenotype in bsd2Δ/rer1Δ yeast. This is the first description of a yeast complementation assay for Slc11a1 function. Given the prior demonstration in frog oocytes that Slc11a1 acts as an antiporter, the most plausible interpretation of the data is that Slc11a1 is rescuing bsd2Δ/rer1Δ yeast by exporting divalent cations. Chimaeras define the N terminus, and a segment of the protein core preceding transmembrane domain 9 through transmembrane domain 12, as important in rescuing the divalent cation stress phenotype. EGTA sensitivity and divalent cation stress phenotypes in yeast expressing Slc11a orthologues show that symport activity is ancestral. Molecular changes that mediate rescue of the divalent cation stress phenotype post-date frogs and co-evolved with Slc11a1 orthologues that regulate divalent cation homeostasis in macrophages and resistance to infection in chickens and mammals.

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pH-Induced Conformational Changes inClostridium difficile Toxin B

Infection and Immunity ◽

10.1128/iai.68.5.2470-2474.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 2470-2474 ◽

Cited By ~ 111

Author(s):

Maen Qa'Dan ◽

Lea M. Spyres ◽

Jimmy D. Ballard

Keyword(s):

Conformational Changes ◽

Mammalian Cells ◽

Structural Changes ◽

Tryptophan Fluorescence ◽

Acidic Ph ◽

Bafilomycin A1 ◽

Toxin B ◽

Endosomal Acidification ◽

Specific Protease ◽

The Impact

ABSTRACT Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.

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Infectivity of dengue virus serotypes 1 and 2 is correlated to E protein intrinsic dynamics but not to envelope conformations

10.1101/303560 ◽

2018 ◽

Author(s):

Kamal Kant SHARMA ◽

Xin-Xiang LIM ◽

Sarala Neomi TANTIRIMUDALIGE ◽

Anjali Gupta ◽

Jan K MARZINEK ◽

...

Keyword(s):

Structural Dynamics ◽

Conformational Changes ◽

Large Scale ◽

Morphological Changes ◽

Divalent Cations ◽

Virulent Strain ◽

Viral Envelope ◽

Temperature Dependent ◽

Dengue Virus Serotypes ◽

Dynamics Simulations

Dengue is a mosquito-borne virus with dire health and economic impact. Dengue is responsible for an estimated ~390 million infections per year, with Dengue 2 (DENV2) being the most virulent strain among the four serotypes. Interestingly, it is also for strains of this serotype that temperature-dependent large scale morphological changes, termed as 'breathing', have been observed. Although, the structure of these morphologies has been solved to 3.5 Angstrom resolution, the dynamics of the viral envelope are unknown. Here, we combine fluorescence and mass spectrometry and molecular dynamics simulations to provide insights into DENV2 structural dynamics in comparison to DENV1. We observe hitherto unseen conformational changes and structural dynamics of the DENV2 envelope that are influenced by both temperature and divalent cations. Our results show that for DENV2 and DENV1 the intrinsic dynamics but not the specific morphologies are correlated to viral infectivity.

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The extracellular matrix–myosin pathway in mechanotransduction: from molecule to tissue

Emerging Topics in Life Sciences ◽

10.1042/etls20180043 ◽

2018 ◽

Vol 2 (5) ◽

pp. 727-737 ◽

Cited By ~ 2

Author(s):

Ionel Popa ◽

Jennifer H. Gutzman

Keyword(s):

Extracellular Matrix ◽

Cell Migration ◽

Conformational Changes ◽

Molecular Mechanisms ◽

Morphological Changes ◽

Specific Binding ◽

Mechanical Stimuli ◽

Tissue Architecture ◽

Mechanical Signaling ◽

Cellular Development

Mechanotransduction via the extracellular matrix (ECM)–myosin pathway is involved in determining cell morphology during development and in coupling external transient mechanical stimuli to the reorganization of the cytoskeleton. Here, we present a review on the molecular mechanisms involved in this pathway and how they influence cellular development and organization. We investigate key proteins involved in the ECM–myosin pathway and discuss how specific binding events and conformational changes under force are related to mechanical signaling. We connect these molecular mechanisms with observed morphological changes at the cellular and organism level. Finally, we propose a model encompassing the biomechanical signals along the ECM–myosin pathway and how it could be involved in cell adhesion, cell migration, and tissue architecture.

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Structural basis for activation and gating of IP3 receptors

10.1101/2021.10.18.464806 ◽

2021 ◽

Author(s):

Emily A Schmitz ◽

Hirohide Takahashi ◽

Erkan Karakas

Keyword(s):

Conformational Changes ◽

Molecular Mechanisms ◽

Receptor Activation ◽

Structural Basis ◽

Ip3 Receptors ◽

Cellular Processes ◽

Cryo Electron Microscopy ◽

Channel Opening ◽

High Concentrations ◽

Type 3

Calcium (Ca2+) is a universal and versatile cellular messenger used to regulate numerous cellular processes in response to external or internal stimuli. A pivotal component of the Ca2+ signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), which mediate Ca2+ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca2+ concentrations. IP3Rs are activated by IP3 and Ca2+, inhibited by Ca2+ at high concentrations, and potentiated by ATP1-3. However, the underlying molecular mechanisms are unclear due to the lack of structures in the active conformation. Here we report cryo-electron microscopy (cryo-EM) structures of human type-3 IP3R in multiple gating conformations; IP3-ATP bound pre-active states with closed channels, IP3-ATP-Ca2+ bound active state with an open channel, and IP3-ATP-Ca2+ bound inactive state with a closed channel. The structures demonstrate how IP3-induced conformational changes prime the receptor for activation by Ca2+, how Ca2+ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism of the receptor activation and gating.

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Effect of verapamil on cytoplasmic distribution of granules and microfilaments in amphibian urinary bladder

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103681 ◽

1988 ◽

Vol 46 ◽

pp. 324-325

Author(s):

A.J. Mia ◽

L.X. Oakford ◽

T. Yorio

Keyword(s):

Urinary Bladder ◽

Water Flow ◽

Morphological Changes ◽

Granular Cell ◽

Cytosolic Calcium ◽

Calcium Storage ◽

Amphibian Urinary Bladder ◽

Vesicular Membrane ◽

High Concentrations ◽

Cytoskeletal System

The amphibian urinary bladder has been used as a ‘model’ system for studies of the mechanism of action of antidiuretic hormone (ADH) in stimulating transepithelial water flow. The increase in water permeability is accompanied by morphological changes that include the stimulation of apical microvilli, mobilization of microtubules and microfilaments and vesicular membrane fusion events . It has been shown that alterations in the cytosolic calcium concentrations can inhibit ADH transmembrane water flow and induce alterations in the epithelial cell cytomorphology, including the cytoskeletal system . Recently, the subapical granules of the granular cell in the amphibian urinary bladder have been shown to contain high concentrations of calcium, and it was suggested that these cytoplasmic constituents may act as calcium storage sites for intracellular calcium homeostasis. The present study utilizes the calcium antagonist, verapamil, to examine the effect of calcium deprivation on the cytomorphological features of epithelial cells from amphibian urinary bladder, with particular emphasis on subapical granule and microfilament distribution.

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Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157760 ◽

1990 ◽

Vol 48 (3) ◽

pp. 44-45

Author(s):

G-A. Keller ◽

S. J. Gould ◽

S. Subramani ◽

S. Krisans

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Firefly Luciferase ◽

Peroxisomal Enzyme ◽

Transfected Cells ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Series Of Experiments ◽

Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

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