Thermodynamic Dissection of a Low Affinity Protein-Protein Interface Involved in Human Immunodeficiency Virus Assembly

Álamo, Marta del; Neira, José L.; Mateu, Mauricio G.

doi:10.1074/jbc.m304466200

Cited by 60 publications

(112 citation statements)

References 51 publications

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“…2), which harbors the dimerization domain, to the C-terminal end of MA. Two nearly identical versions of this protein were constructed, which either abrogate dimerization (W184A, M185A; here called MA-CTDmon) or enhance dimerization by de- creasing the K D by 10-fold (Q192A; here called MA-CTDdim, (12). Proteins similar to these, but based on RSV MA instead of HIV-1 MA, previously were verified by velocity sedimentation to be monomeric or dimeric, respectively (10).…”

Section: Resultsmentioning

confidence: 99%

Electrostatic Interactions Drive Membrane Association of the Human Immunodeficiency Virus Type 1 Gag MA Domain

Dalton

Ako-adjei

Murray

et al. 2007

J Virol

106

152

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The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids.

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Section: Resultsmentioning

confidence: 99%

Electrostatic Interactions Drive Membrane Association of the Human Immunodeficiency Virus Type 1 Gag MA Domain

Dalton

Ako-adjei

Murray

et al. 2007

J Virol

106

152

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“…The HIV-1 CTD is known to selfassociate with a moderate affinity (K D , ϳ10 Ϫ5 M) (16,23,76). This self-association can be abrogated by changing two hydrophobic amino acids at the dimer interface (W184A and M185A) and can be enhanced by an order of magnitude (K D , ϳ10 Ϫ6 M) by mutating a glutamine residue (Q192A) (16). We incorporated both sets of mutations into fusion proteins, creating MACA CTD(W184A/M185A) and MACA CTD(Q192A) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of Rous Sarcoma Virus MA Protein Interaction with Membranes

Dalton

Murray

et al. 2005

J Virol

108

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The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo.

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“…52 Nuclear magnetic resonance Solid state NMR experiments on both uniformly 15 N, 13 C-labeled and 1,3-13 C 2 -glycerol-labeled CA assemblies were performed with a Varian InfinityPlus spectrometer operating at 150.7 MHz 13 C NMR frequency and a Varian triple-channel balun MAS NMR probe with 3.2 mm rotors. Sample heating by rf irradiation was significant, due to the 1M NaCl concentration in CA assembly samples, requiring that cooling gas at À80 C be supplied to the variable-temperature stack of the NMR probe to maintain sample temperatures in the [30][31][32][33][34][35][36][37][38][39][40] C range. The temperature of the sample was higher than that of the cooling gas both because the cooling gas warmed by $50 C while passing through the variable-temperature stack and because the rf irradiation heated the sample directly during measurements.…”

Section: Electron Microscopymentioning

confidence: 99%

“…1 The capsid is believed to be formed by a triangular lattice of CA hexamers, with 12 CA pentamers unevenly distributed at the two ends of the cone to produce a closed shell. 2 Although much progress has been made toward understanding the structure, stability, and assembly of the HIV-1 capsid, through electron microscopy, [3][4][5][6][7][8][9][10] X-ray crystallography, [11][12][13][14][15][16][17][18][19][20] solution nuclear magnetic resonance (NMR), 13,[21][22][23][24][25][26][27] and various biophysical techniques, [28][29][30][31][32][33][34][35][36][37][38][39][40] substantial questions remain regarding the details of intermolecular interactions within and between CA hexamers, the structural variations that permit CA to form both hexamers and pentamers, the driving force for curvature and closure of the CA lattice, and the mechanism and pathway for CA self-assembly. In principle, modern solid state NMR techniques, which can provide sitespecific molecular-level structural information about noncrystalline protein assemblies, [41][...…”

Section: Introductionmentioning

confidence: 99%

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

Chen

Tycko

2010

Protein Science

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The wild-type HIV-1 capsid protein (CA) self-assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV-1. Mass-per-length values of CA assemblies determined by dark-field transmission electron microscopy indicate a variety of structures, ranging from single-wall tubes to multiwall tubes that approximate solid rods. Two-dimensional (2D) solid state 13 CA 13 C and 15 NA 13 C NMR spectra of uniformly 15 N, 13 C-labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3-13 C 2 -glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N-terminal (NTD) and C-terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self-assembly of full-length CA into tubes. 2D 1 H-13 C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the a-helices of NTD and CTD and from the N-and C-terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.

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Thermodynamic Dissection of a Low Affinity Protein-Protein Interface Involved in Human Immunodeficiency Virus Assembly

Cited by 60 publications

References 51 publications

Electrostatic Interactions Drive Membrane Association of the Human Immunodeficiency Virus Type 1 Gag MA Domain

Electrostatic Interactions Drive Membrane Association of the Human Immunodeficiency Virus Type 1 Gag MA Domain

Biochemical Characterization of Rous Sarcoma Virus MA Protein Interaction with Membranes

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

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