Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

Efficient assembly of HIV-1 at the plasma membrane (PM) of the T-cell specifically requires PI(4,5)P 2 . It was previously shown that a highly basic region (HBR) of the matrix protein (MA) on the Gag precursor polyprotein Pr55 Gag is required for membrane association. MA is N-terminally myristoylated, which enhances its affinity to membranes. In this work we used X-ray scattering and neutron reflectivity to determine how the physical properties and structure of lipid bilayers respond to the addition of binding domain peptides, either in the myristoylated form (MA 31 myr) or without the myristoyl group (MA 31 ). Neutron reflectivity measurements showed the peptides predominantly located in the hydrocarbon interior. Diffuse X-ray scattering showed differences in membrane properties upon addition of peptides and the direction of the changes depended on lipid composition. The PI(4,5)P 2 -containing bilayers softened, thinned and became less ordered as peptide concentration increased. In contrast, POPS-containing bilayers with equivalent net charge first stiffened, thickened and became more ordered with increasing peptide concentration. As softening the host cell's PM upon contact with the protein lowers the free energy for membrane restructuring, thereby potentially facilitating budding of viral particles, our results suggest that the role of PI(4,5)P 2 in viral assembly goes beyond specific stereochemical membrane binding. These studies reinforce the importance of lipids in virology.

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“…Our interpretation may seem contrary to published neutron reflectivity results [6,55]. However, Ref.…”

Section: Location Of the Peptides In The Membrane Mimicscontrasting

confidence: 99%

“…However, Ref. [55] used NR to study the non-myristoylated MA protein which was positioned at the surface of a bilayer containing saturated lipids. The results in Ref.…”

Section: Location Of the Peptides In The Membrane Mimicsmentioning

confidence: 99%

HIV-1 Matrix-31 Membrane Binding Peptide Interacts Differently with Membranes Containing PS vs. PI(4,5)P 2

O'neil

Andenoro

Pagano

et al. 2017

Biophysical Journal

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“…The SPR responses at high c p were accordingly corrected. From a calibration of the SPR instrument (37), we estimate that a densely packed monolayer of MA protein corresponds to an instrument response of R ϱ Ϸ 58 pixels, as determined from its molecular cross section (A ϭ 786 Å 2 ), assessed from the nuclear magnetic resonance (NMR) structure (26), in the membranebound orientation (38) and the molecular weight. Free binding energies (⌬G) were then calculated from the K d value:…”

Section: Methodsmentioning

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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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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“…The following observations suggest that this is not the case. 1) Structural investigations with NR of other proteins at membrane surfaces, conducted at similar protein surface concentrations (21,34,38), showed clearly that protein organization depended on other factors (electrostatic interaction; ligand binding) than surface crowding. 2) A model that places proteins randomly on the surface (39) predicts mean nearest neighbor distances of 1.6 or 2.25 protein diameters, respectively, for area coverages of 20% (myrG55) or 10% (G55).…”

Section: Structure Of the Grasp Domainmentioning

confidence: 98%

“…Complex with the Membrane-To correlate the functional requirements for membrane tethering with structural details of the GRASP domain complex with a membrane, we studied the latter with NR, a technique that can determine the orientation of membranebound proteins of sufficient shape anisotropy within tight limits (34) and their localization along the membrane normal with Angstrom precision (24 O differ in neutron scattering lengths, regions of the surface structure that contain water contribute differently to the scattering in isotopically distinct buffers, thereby facilitating the characterization of such regions within the sample. Neutron scattering is nondestructive, which allows all measurements to be conducted on the same sample area under identical conditions (19,35).…”

Section: Structure Of the Grasp Domainmentioning

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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Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

Cited by 48 publications

References 44 publications

HIV-1 Matrix-31 Membrane Binding Peptide Interacts Differently with Membranes Containing PS vs. PI(4,5)P 2

HIV-1 Matrix-31 Membrane Binding Peptide Interacts Differently with Membranes Containing PS vs. PI(4,5)P 2

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

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

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