H+-induced Membrane Insertion of Influenza Virus Hemagglutinin Involves the HA2 Amino-terminal Fusion Peptide but Not the Coiled Coil Region

Durrer, Peter; Galli, Carmela; Hoenke, Stefan; Corti, Chantal; Glück, Reinhard; Vorherr, Thomas; Brunner, Josef

doi:10.1074/jbc.271.23.13417

Cited by 197 publications

(130 citation statements)

References 28 publications

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“…The longest peptide consisted of 20 guest residues, because mostly hydrophilic residues follow the first 20 residues of influenza HA 2 ; these subsequent residues do not insert into lipid bilayers as was demonstrated by photoaffinity labeling (2). Good solubility in aqueous buffers was achieved by linking the fusion peptides to a very polar host peptide.…”

Section: Resultsmentioning

confidence: 99%

“…Fusion peptides are highly conserved and quite hydrophobic sequences of fusion proteins that reside in viral envelope membranes or in the plasma membranes of sperm cells (1). These peptides are thought to be the only segments of the fusion proteins that insert deeply into the lipid bilayer of the target membrane at an early stage of membrane fusion (2). Despite this prominent role and numerous biophysical studies, no precise structure of a fusion peptide in lipid bilayers has yet been determined.…”

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confidence: 99%

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A host–guest system to study structure–function relationships of membrane fusion peptides

Han

Tamm²

2000

Proc. Natl. Acad. Sci. U.S.A.

135

158

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We designed a host-guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis. They undergo a conformational change from random coil to an obliquely inserted (Ϸ33°from the surface) ␣-helix on binding to model membranes. Partition coefficients for membrane insertion were measured for influenza fusion peptides of increasing lengths (n ‫؍‬ 8, 13, 16, and 20). The hydrophobic contribution to the free energy of binding of the 20-residue fusion peptide at pH 5.0 is ؊7.6 kcal͞mol (1 cal ‫؍‬ 4.18 J). This energy is sufficient to stabilize a ''stalk'' intermediate if a typical number of fusion peptides assemble at the site of membrane fusion. The fusion activity of the fusion peptides increases with each increment in length, and this increase strictly correlates with the hydrophobic binding energy and the angle of insertion.F usion peptides play a central role in facilitating membrane fusion. Fusion peptides are highly conserved and quite hydrophobic sequences of fusion proteins that reside in viral envelope membranes or in the plasma membranes of sperm cells (1). These peptides are thought to be the only segments of the fusion proteins that insert deeply into the lipid bilayer of the target membrane at an early stage of membrane fusion (2). Despite this prominent role and numerous biophysical studies, no precise structure of a fusion peptide in lipid bilayers has yet been determined. Also, the binding of fusion peptides to lipid bilayers could not be studied previously, because these peptides are too hydrophobic to measure their partitioning between aqueous and membrane phases. Obtaining thermodynamic parameters of fusion peptide binding to lipid bilayers would be extremely helpful for the formulation of physically relevant models of energy coupling in membrane fusion. Most previous physical studies of fusion peptide-bilayer interactions were conducted with samples in which peptides and lipids were mixed in organic solvents and hydrated or added from organic solvent to preformed liposomes before spectroscopic analysis. A notable exception was a spin-label EPR spectroscopic study in which an expressed fragment of influenza hemagglutinin (HA) was used to assess the depth of the fusion peptide in lipid bilayers (3). The conformation of hydrophobic peptides in lipid bilayers often depends on the solvent that was used in the reconstitution procedure. Gramicidin A, for example, adopts many different conformations depending on how the samples are prepared (4). Therefore, the current procedures to reconstitute fusion peptide-bilayer complexes may not be adequate for higher-resolution structural studies of fusion peptides in lipid bilayers. To alleviate these problems, we ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

A host–guest system to study structure–function relationships of membrane fusion peptides

Han

Tamm²

2000

Proc. Natl. Acad. Sci. U.S.A.

135

158

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“…[ 125 I]TID-PC/16 has previously been used to identify and characterize regions within membrane proteins that interact with lipids (11,14). Its physicochemical behavior in terms of mobility in thin layer chromatography is indistinguishable from PC, and its interaction with the transmembrane region of integral membrane proteins also appears to be identical to that of PC (12,13).…”

Section: Discussionmentioning

confidence: 99%

“…This reagent partitions in the phospholipidic milieu and upon photolysis reacts indiscriminately with its molecular environment. It is thus possible to directly analyze the interaction between the hydrophobic membrane-spanning domain of a membrane protein and lipids belonging to its immediate environment (12)(13)(14). Applying this technique on the PMCA, we were able to measure equilibrium constants for the dissociation of ligands from PMCA complexes and to draw structural conclusions about the regulation of the transport of Ca 2ϩ in the presence of different modulators.…”

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confidence: 98%

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

Mangialavori

Ferreira-Gomes

Pignataro

et al. 2010

Journal of Biological Chemistry

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The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca 2؉ -pump (PMCA) in the membrane region upon interaction with Ca 2؉ , calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125 I]TID-PC/16, measuring the shift of conformation E 2 to the auto-inhibited conformation E 1 I and to the activated E 1 A state, titrating the effect of Ca 2؉ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca 2؉ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca 2؉ transport but does not modify the affinity for Ca 2؉ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E 1 I to the activated E 1 A conformation and thus modulating the transport of Ca 2؉ . This is reflected in the different apparent constants for Ca 2؉ in the absence of CaM (calculated by Ca 2؉ -ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca 2؉ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca 2؉ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca 2؉ binding and activation.Detailed structural information about the plasma membrane calcium pump (PMCA) 2 is currently lacking. This pump is an integral part of the Ca 2ϩ signaling mechanism (1) and is thus a crucial component of cell function. It is highly regulated by calmodulin (CaM), which activates this protein by binding to an auto-inhibitory region (2) and changes the conformation of the pump from an inhibited state to an activated one (2, 3).Information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. In this work, we have used a hydrophobic photolabeling method to study the noncovalent interactions between the membrane domain of the PMCA and surrounding phospholipids under different experimental conditions that lead to known conformations. It has been previously demonstrated that both the conformation and the activity of the pump protein are preserved in either solubilized or reconstituted purified preparations compared with the native pump located in the erythrocyte (4).In recent work, we assessed changes in the overall exposure of PMCA to surrounding lipids by quantifying the extent of protein labeling by the photoactivatable phosphatidylcholine analog [125 I]TID-PC/16 under different conditions (5). This showed that labeling of ...

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“…However, in the case of HIV-1 gp41 fusion peptide insertion has yet to be demonstrated. This has been done for influenza hemagglutinin (37) and VSV (38) by the group of Joseph Brunner, the pioneer of hydrophobic membrane protein photolabeling techniques, using carbene-based hydrophobic markers incorporated in liposomes. Liposomes are, however, not suitable targets for HIV, and our labeling approach is based on the use of aryl azides that can be added to viruses or cells and activated in situ.…”

Section: Discussionmentioning

confidence: 99%

Photoinduced Reactivity of the HIV-1 Envelope Glycoprotein with a Membrane-Embedded Probe Reveals Insertion of Portions of the HIV-1 Gp41 Cytoplasmic Tail into the Viral Membrane

et al. 2008

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The interactions of HIV-1 Env (gp120-gp41) with CD4 and coreceptors trigger a barrage of conformational changes in Env that drive the membrane fusion process. Various regions of gp41 have profound effects on HIV entry and budding. However, the precise interactions between gp41 and the membrane have not been elucidated. To examine portions of membrane proteins that are embedded in membrane lipids, we have studied photoinduced chemical reactions in membranes using the lipid bilayer specific probe iodonaphthyl azide (INA). Here we show that in addition to the transmembrane anchor, amphipatic sequences in the cytoplasmic tail (CT) of HIV-1 gp41 are labeled by INA. INA labeling of the HIV-1 gp41 CT was similar whether wild-type or a mutant HIV-1 was used with uncleaved p55 Gag, which does not allow entry. These results shed light on the disposition of the HIV-1 gp41 CT with respect to the membrane. Moreover, our data have general implications for topology of membrane proteins and their in situ interactions with the lipid bilayer.The human immunodeficiency virus (HIV 1 ) gains entry into its target cell through a fusion process driven by its membrane protein gp41 (1). HIV-1 Env (gp120-gp41) is present on the † This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, by a grant from the NIH Intramural AIDS Targeted Antiviral Program (IATAP), and by a grant from the NIAID Intramural Biodefense Research Program. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400. INA is composed of three moieties. The core of the compound is a naphthalene ring that confers a very high hydrophobicity. Its partition coefficient in biological membranes of ∼10 5 (5) ensures its selective and exclusive targeting to the lipidic bilayer. Its azido moiety allows its photoactivation. Upon irradiation with near UV light, the azido group undergoes transformation into a highly reactive nitrene that will covalently bind the proteins and lipids in its vicinity (6). Finally, the iodine on this compound provides a very sensitive detection through its radioactive form. [ 125 I]-INA has been extensively used to exclusively label the portions of membrane proteins that are embedded within the lipid domain (6-13). We applied hydrophobic labeling to the HIV-1 Env expressed on the virus or on the surface of cells to assess the topology of the Env glycoprotein. MATERIALS AND METHODS Reagents and CellsDulbecco's modified Eagle medium (DMEM), RPMI, fetal bovine serum (FBS), G418, hygromycin, and antibiotics were obtained from Invitrogen (Carlsbad, CA). 293T and HeLa cells were cultured in DMEM supplemented with 10% FBS and antibiotics (DMEM-10). Different HIV-1 Env were transiently expressed on the surface of HeLa cells using the recombinant vaccinia vectors vSC60 (IIIB/Lai BH8 Env) or vPE17 (14) (IIIB/Lai BH8 Env truncated at AA733). GHOST (3) X4/R5 (G345), a HOS...

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H+-induced Membrane Insertion of Influenza Virus Hemagglutinin Involves the HA2 Amino-terminal Fusion Peptide but Not the Coiled Coil Region

Abstract: Fusion of influenza virus with target membranes is

Cited by 197 publications

References 28 publications

A host–guest system to study structure–function relationships of membrane fusion peptides

A host–guest system to study structure–function relationships of membrane fusion peptides

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

Photoinduced Reactivity of the HIV-1 Envelope Glycoprotein with a Membrane-Embedded Probe Reveals Insertion of Portions of the HIV-1 Gp41 Cytoplasmic Tail into the Viral Membrane

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