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

2016 ◽  
Vol 90 (9) ◽  
pp. 4544-4555 ◽  
Author(s):  
Marilia Barros ◽  
Frank Heinrich ◽  
Siddhartha A. K. Datta ◽  
Alan Rein ◽  
Ioannis Karageorgos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Matrix Protein ◽  
Full Length ◽  
Protein Protein Interactions ◽  
Binding Studies ◽  
Protein Protein Interaction ◽  
Gag Polyprotein ◽  
Protein Lipidation ◽  
Hiv 1

ABSTRACTBy 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, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2attract 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)P2trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, 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)P2binding. 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.

scholarly journals A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions

2004 ◽  
Vol 78 (3) ◽  
pp. 1230-1242 ◽  
Author(s):  
Aaron Derdowski ◽  
Lingmei Ding ◽  
Paul Spearman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55Gag, we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.

scholarly journals PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1895
Author(s):  
Alžběta Dostálková ◽  
Kryštof Škach ◽  
Filip Kaufman ◽  
Ivana Křížová ◽  
Romana Hadravová ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Early Stage ◽  
Protein A ◽  
Protein Protein Interactions ◽  
Particle Assembly ◽  
Gag Polyprotein ◽  
Polyprotein Precursor ◽  
Hiv 1 ◽  
Ca Protein

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein–protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

scholarly journals Human Immunodeficiency Virus Type 1 Gag Polyprotein Multimerization Requires the Nucleocapsid Domain and RNA and Is Promoted by the Capsid-Dimer Interface and the Basic Region of Matrix Protein

1999 ◽  
Vol 73 (10) ◽  
pp. 8527-8540 ◽  
Author(s):  
Mark T. Burniston ◽  
Andrea Cimarelli ◽  
John Colgan ◽  
Sean P. Curtis ◽  
Jeremy Luban
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Protein Protein Interactions ◽  
Virion Assembly ◽  
Dimer Interface ◽  
Gag Polyprotein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Manygag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed intrans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.

scholarly journals Small-molecule CaVα1⋅CaVβ antagonist suppresses neuronal voltage-gated calcium-channel trafficking

2018 ◽  
Vol 115 (45) ◽  
pp. E10566-E10575 ◽  
Author(s):  
Xingjuan Chen ◽  
Degang Liu ◽  
Donghui Zhou ◽  
Yubing Si ◽  
David Xu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Calcium Channel ◽  
Small Molecule ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Channel Trafficking ◽  
Voltage Gated ◽  
Protein Protein Interaction ◽  
Computational Screening

Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1subunit (CaVα1) that is engaged in protein–protein interactions with auxiliary α2/δ and β subunits. The high-affinity CaV2.2α1⋅CaVβ3protein–protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVβ3protein–protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1. Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVβ3Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein–protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.

Screening Assay for the Detection of the Protein-Protein Interaction Between HIV-1 Nef Protein and the SH3 Domain of Hck

1998 ◽  
Vol 3 (1) ◽  
pp. 37-41 ◽  
Author(s):  
Anne E. Jones ◽  
Kalle Saksela ◽  
Stephen M. Game ◽  
Gerard O'Beirne ◽  
Neil D. Cook
Keyword(s):  
Protein Interactions ◽  
Sh3 Domain ◽  
Protein Protein Interactions ◽  
Screening Assay ◽  
Native Form ◽  
Protein Protein Interaction ◽  
Src Homology ◽  
Equilibrium Dissociation ◽  
High Degree ◽  
Hiv 1

The interaction between the Human Immunodeficiency Virus Nef protein (HIV-1 Nef) and the Src Homology Region 3 (SH3) domain of Hck was studied using scintillation proximity assay (SPA). SPA is a quick and sensitive method that does not require a separation step, thus allowing assays to be performed in a homogeneous environment. In contrast to most conventional techniques, SPA may also be used to detect low affinity protein-protein interactions. In this study, the assay was configured using biotinylated Hck SH3 domain expressed both as a GST fusion protein and synthesized chemically in its' native form. Biotinylated Hck protein was immobilized to streptavidin-coated fluoromicrosphere SPA beads and the binding of [3H]Nef was detected by scintillation counting. Analysis of binding yielded an average equilibrium dissociation constant (KD) of 183 ± 30 nM for the interaction in line with reported values by other methods. The data presented demonstrates that using SPA, protein-protein interactions of relatively low affinity can be detected with a high degree of sensitivity and screening studies of inhibitors of these associations could be facilitated by the high sample throughput achievable with SPA.

scholarly journals Sulfotyrosine, an interaction specificity determinant for extracellular protein-protein interactions

2021 ◽  
Author(s):  
Valley Stewart ◽  
Pamela C. Ronald
Keyword(s):  
Protein Interactions ◽  
Chemokine Receptors ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Protein Protein Interaction ◽  
Coreceptor Binding ◽  
Receptor Interactions ◽  
Technical Advances ◽  
Affinity Peptide ◽  
Hiv 1

Tyrosine sulfation, a post-translational modification, can enhance and often determine protein-protein interaction specificity. Sulfotyrosyl residues (sTyr) are formed by tyrosyl-protein sulfotransferases (TPSTs) during maturation of certain secreted proteins. Here we consider three contexts for sTyr function. First, a single sTyr residue is critical for high-affinity peptide-receptor interactions in plant peptide hormones and animal receptors for glycopeptide hormones. Second, structurally flexible anionic segments often contain a cluster of two or three sTyr residues within a six-residue span. These sTyr residues are essential for coreceptor binding of the HIV-1 envelope spike protein during virus entry and for chemokine interactions with many chemokine receptors. Third, several proteins that interact with thrombin, central to normal blood-clotting, require the presence of sTyr residues in the context of acidic sequences termed hirudin-like motifs. Consequently, many proven and potential therapeutic proteins derived from blood-consuming invertebrates depend on sTyr residues for their activity. Technical advances in generating and documenting site-specific sTyr substitutions facilitate discovery and analysis, and promise to enable engineering of defined interaction determinants.

An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Yu-Miao Zhang ◽  
Jun Wang ◽  
Tao Wu
Keyword(s):  
Subcellular Localization ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Epidermal Cells ◽  
Cyclin Dependent Kinase ◽  
Protein Subcellular Localization ◽  
Protein Protein Interactions ◽  
Efficient System ◽  
Protein Protein Interaction ◽  
Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

Decoding Protein-protein Interactions: An Overview

2020 ◽  
Vol 20 (10) ◽  
pp. 855-882
Author(s):  
Olivia Slater ◽  
Bethany Miller ◽  
Maria Kontoyianni
Keyword(s):  
Drug Discovery ◽  
Protein Interactions ◽  
Drug Repurposing ◽  
Protein Docking ◽  
Target Space ◽  
Protein Protein Interactions ◽  
X Ray Crystallography ◽  
Protein Protein Interaction ◽  
Interaction Sites ◽  
Long Time

Drug discovery has focused on the paradigm “one drug, one target” for a long time. However, small molecules can act at multiple macromolecular targets, which serves as the basis for drug repurposing. In an effort to expand the target space, and given advances in X-ray crystallography, protein-protein interactions have become an emerging focus area of drug discovery enterprises. Proteins interact with other biomolecules and it is this intricate network of interactions that determines the behavior of the system and its biological processes. In this review, we briefly discuss networks in disease, followed by computational methods for protein-protein complex prediction. Computational methodologies and techniques employed towards objectives such as protein-protein docking, protein-protein interactions, and interface predictions are described extensively. Docking aims at producing a complex between proteins, while interface predictions identify a subset of residues on one protein that could interact with a partner, and protein-protein interaction sites address whether two proteins interact. In addition, approaches to predict hot spots and binding sites are presented along with a representative example of our internal project on the chemokine CXC receptor 3 B-isoform and predictive modeling with IP10 and PF4.

scholarly journals Short loop functional commonality identified in leukaemia proteome highlights crucial protein sub-networks

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Sun Sook Chung ◽  
Joseph C F Ng ◽  
Anna Laddach ◽  
N Shaun B Thomas ◽  
Franca Fraternali
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Interaction Network ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Ppi Networks ◽  
Short Loop ◽  
New Strategy ◽  
Loop Network ◽  
Protein Protein Interaction Network

Abstract Direct drug targeting of mutated proteins in cancer is not always possible and efficacy can be nullified by compensating protein–protein interactions (PPIs). Here, we establish an in silico pipeline to identify specific PPI sub-networks containing mutated proteins as potential targets, which we apply to mutation data of four different leukaemias. Our method is based on extracting cyclic interactions of a small number of proteins topologically and functionally linked in the Protein–Protein Interaction Network (PPIN), which we call short loop network motifs (SLM). We uncover a new property of PPINs named ‘short loop commonality’ to measure indirect PPIs occurring via common SLM interactions. This detects ‘modules’ of PPI networks enriched with annotated biological functions of proteins containing mutation hotspots, exemplified by FLT3 and other receptor tyrosine kinase proteins. We further identify functional dependency or mutual exclusivity of short loop commonality pairs in large-scale cellular CRISPR–Cas9 knockout screening data. Our pipeline provides a new strategy for identifying new therapeutic targets for drug discovery.

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