Structure and Properties of Tethered Bilayer Lipid Membranes with Unsaturated Anchor Molecules

Budvytytė, Rima; Valinčius, Gintaras; Niaura, Gediminas; Voiciuk, Vladislava; Mickevicius, Mindaugas; Chapman, Hilary; Goh, Haw-Zan; Shekhar, Prabhanshu; Heinrich, Frank; Shenoy, Siddharth; Lösche, Mathias; Vanderah, David J.

doi:10.1021/la401132c

Cited by 102 publications

(130 citation statements)

References 59 publications

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“…The plasmid used for the ϩmyrMA preparation via coexpression of (31). Lipid mixtures at the desired molar ratios were prepared from fresh lipid stock in chloroform, and the solvent was evaporated under vacuum for 12 h. The dried lipid films were hydrated in a high-salt aqueous buffer (1 M NaCl, 10 mM NaPO 4 [pH 7.4]) to a lipid concentration of ϳ5 mg/ml and subsequently sonicated until clear lipid solutions were obtained.…”

Section: Methodsmentioning

confidence: 99%

“…1) incorporates a single lipidic bilayer membrane of wellcontrolled composition that is tethered to a planar electrode-a 0.05-m-thick Au film on a glass or Si substrate-via short ethyleneoxide anchors (31), thereby providing a hydrated environment on both sides of the membrane. Thereby, the bilayer retains its intrinsic fluidity (32), is virtually defect-free (29), and is resilient to external manipulations such as buffer exchanges, as monitored by electrochemical impedance spectroscopy (33).…”

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confidence: 99%

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Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

Barros

Heinrich

Datta

et al. 2016

J Virol

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By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals-electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ⌬G, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P 2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P 2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P 2 , the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P 2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCELike other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. T he polyprotein Gag is an essential component for the reproduction of retroviruses, such as HIV-1, in infected cells. In the production of new viruses, many copies of Gag assemble laterally, either in the cytoplasm or on the plasma membrane (PM) of the host, thus acquiring their lipid envelope as they bud from the cell. Distinct retroviral genera encode Gag proteins which all show a similar domain architecture, with an N-terminal matrix (MA) domain that targets the PM, followed by the capsid (CA) and nucleocapsid ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

Barros

Heinrich

Datta

et al. 2016

J Virol

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“…Both the SPR method for quantifying intermolecular interactions (51) and the sparsely tethered bilayer lipid membrane (stBLM) model membrane system (52) are well established. stBLMs incorporate a single lipidic bilayer membrane of controlled composition that is tethered to a planar electrode-a 0.05-m-thick Au film on a glass or Si substrate-via short ethylene oxide anchors (53), thereby providing a hydrated environment on both sides of the membrane. Thus, the bilayer retains its intrinsic fluidity (54), is virtually defect free (52), and is resilient to external manipulations, such as buffer exchanges, as monitored by electrochemical impedance spectroscopy (EIS) (55).…”

Section: Methodsmentioning

confidence: 99%

Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

Dick

Barros

Jin

et al. 2016

J Virol

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The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity. L ate in the retroviral life cycle, the structural protein Gag localizes to the inner leaflet of the plasma membrane (PM), where it assembles around the viral genomic RNA, leading to budding from the cell. Gag proteins include three major domains, the Nterminal matrix (MA) domain, which mediates membrane binding; the central capsid (CA) domain, which mediates Gag-Gag interactions during virion assembly; and the C-terminal nucleocapsid (NC...

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“…The tether lipid for supported membranes, Z 20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol (HC18) (17), was a generous gift from Dr. David Vanderah (Institute for Bioscience and Biotechnology Research, Rockville, MD). ␤-Mercaptoethanol was from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

Heinrich

Nanda

Goh

et al. 2014

Journal of Biological Chemistry

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Background: An unknown mechanism promotes trans interactions by the GRASP homotypic membrane tethers rather than unproductive cis interactions. Results: Neutron reflection shows that the myristoylated GRASP domain has a fixed, upright orientation on the membrane incompatible with cis interactions. Conclusion: Myristoylation restricts the orientation of the protein on the membrane to favor interactions in trans. Significance: Orientation of membrane proteins is functionally significant and may be regulated by myristoylation.

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Structure and Properties of Tethered Bilayer Lipid Membranes with Unsaturated Anchor Molecules

Cited by 102 publications

References 59 publications

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

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