Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties

Doktorova, Milka; Heberle, Frederick A.; Kingston, Richard L.; Khelashvili, George; Cuendet, Michel A.; Wen, Yi; Feigenson, Gerald W.; Vogt, Volker M.; Dick, Robert A.

doi:10.1016/j.bpj.2017.08.055

Cited by 41 publications

(45 citation statements)

References 62 publications

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“…budding but not HIV-1 Gag budding. Residues 18, 67, and 72 in ASV Gag have been implicated in PS binding in studies reported by Doktorova et al (21). The effects on targeting or budding of single (15) or triple substitutions were only partial (this report).…”

Section: Determinants Of Asv Gag Plasma Membrane Targetingsupporting

confidence: 51%

“…This was especially found to be the case when the central MA region was removed. The ASV MA sequence in Gag is highly basic and binds phosphatidylserine-containing membranes through electrostatic interactions (21,(43)(44)(45). Such membrane interaction is blocked by RNA binding to this region (19).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, affecting either lipid could have some effect on ASV Gag. Doktorova et al (21) suggested that Lys 18 , Lys 67 , and Lys 72 might be involved in PS interactions. Their individual contributions to the overall binding were small (51), and Callahan et al observed only a partial effect on particle production with single mutants (15).…”

Section: Mutations In Putative Ma Residues Important For Ps Binding Imentioning

confidence: 99%

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The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery

Watanabe

Medina

Eastep

et al. 2018

Journal of Biological Chemistry

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Edited by Charles E. Samuel The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P 2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P 2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P 2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase C␦ (PH-PLC), two proteins that bind PI(4,5)P 2 , affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P 2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P 2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P 2 is a strong determinant of ASV Gag targeting to the PM and budding. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2) 2 is an essential lipid in eukaryotic cells that functions in many differ

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Section: Determinants Of Asv Gag Plasma Membrane Targetingsupporting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

Section: Mutations In Putative Ma Residues Important For Ps Binding Imentioning

confidence: 99%

See 1 more Smart Citation

The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery

Watanabe

Medina

Eastep

et al. 2018

Journal of Biological Chemistry

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“…During the preparation of this manuscript, Doktorova et al (48) reported the X-ray structure of full-length ASV MA (155 amino acids) at 2.8 Å resolution. In this structure, residues 1-102 were resolved, but there was no interpretable electron density for residues 103-155, consistent with an unstructured conformation.…”

Section: Characterization Of Asv Matrix-membrane Interactionsmentioning

confidence: 99%

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly

Vlach

Eastep

Ghanam

et al. 2018

Journal of Biological Chemistry

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For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA 87) to lipids and liposomes. We report that MA 87 binds to the polar head of phosphoinositides such as PI(4,5)P 2. We found that MA 87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA 87-IP binding is governed by charge-charge interactions. Using a sensitive NMRbased liposome-binding assay, we show that binding of MA 87 to liposomes is enhanced by incorporation of PI(4,5)P 2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA 87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly. Retroviral genomes encode a polyprotein called Gag, which contains all of the structural elements required for virus assembly. Subsequent to their synthesis, Gag proteins for most retroviruses are targeted to the plasma membrane (PM) 2 for assem-This work was supported by National Institutes of Health Grant 5R01 GM117837 (to J. S. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Tables S1 and S2 and Figs. S1-S9. The atomic coordinates and structure factors (codes 6CCJ, 6CE5, 6CUS, 6CV8, and 6CW4) have been deposited in the Protein Data Bank (http://wwpdb.org/). The chemical shift data reported in this paper have been deposited in the Biological Magnetic Resonance Bank with the accession codes 27396 and 30404.

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“…The increase in the polarity of the vesicle surface, which can be clearly observed in Figure S3 (blue and red symbols) in Supplemental material, is in agreement with a recently molecular dynamics calculated cholesterol-driven increase in membrane surface charge density. (Doktorova et al, 2017) On the other hand, the hydrophobic tail buried deep in the bilayer reduces the polarity of the middle bilayer as sensed by the doxyl nitroxide, green and brown symbols Figures 3b and 4b and Figure S4. The change in A + of 16-DSE is about two to three times larger than the change A + of TP, which is a strong indication that cholesterol is located below the polar shell deeper in the bilayer.…”

Section: Resultsmentioning

confidence: 99%

Interaction of the β amyloid – Aβ(25–35) – peptide with zwitterionic and negatively charged vesicles with and without cholesterol

Singh

Perić

2018

Chemistry and Physics of Lipids

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The interactions of the Alzheimer’s β-amyloid peptide, Aβ(25–35), with 18:1 (Δ9-Cis) PC 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), L-α-phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (DOPG), and L-α-phosphatidylglycerol (EPG) phospholipid vesicles with and without cholesterol (Ch) are studied by the nitroxide spin probe electron paramagnetic resonance (EPR) method. Two nitroxide spin probes, 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl hexadecanoate (TP, TEMPO-Palmitate) and 2-Ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxy (16-DSE), are utilized in the study. TEMPO-Palmitate has the reporting EPR moiety located at the top of this spin probe, while 16-DSE has the reporting EPR moiety located at the tail of the spin probe. These two probes enable us to sample the surface and the middle of the phospholipid bilayer, respectively. All EPR measurements are done above the melting points of all four phospholipids when the bilayer is in the liquid crystal phase, the physiologically relevant phase. Due to non-linear spectral line fitting, the EPR spectral parameters are extracted with high precision. The results show that there are two populations of Aβ(25–35) and that one of them is located in the hydrophobic phospholipid layer below the hydrophilic headgroup region. The second population appears to be weakly coupled to the surface of the bilayer. Both hydrophobic and electrostatic interactions affect the insertion of Aβ(25–35) in the bilayer. Also, there is strong evidence for an interaction between cholesterol and Aβ (25–35), which affects the dielectric and dynamic properties of the bilayer.

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Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties

Cited by 41 publications

References 62 publications

The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery

The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly

Interaction of the β amyloid – Aβ(25–35) – peptide with zwitterionic and negatively charged vesicles with and without cholesterol

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