Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions

ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein–protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.

Download Full-text

The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain

BioTechniques ◽

10.2144/000113452 ◽

2010 ◽

Vol 49 (1) ◽

pp. 519-524 ◽

Cited By ~ 9

Author(s):

Andrew Dixon ◽

Carol Lim

Keyword(s):

Protein Interactions ◽

Nuclear Translocation ◽

Coiled Coil ◽

Protein Protein Interactions ◽

Intracellular Protein ◽

Coiled Coil Domain ◽

Translocation Assay

Download Full-text

The Human STAT2 Coiled-Coil Domain Contains a Degron for Zika Virus Interferon Evasion

10.1101/2021.10.01.462781 ◽

2021 ◽

Author(s):

Jean-Patrick Parisien ◽

Jessica J Lenoir ◽

Gloria Alvarado ◽

Curt M Horvath

Keyword(s):

Virus Infection ◽

Protein Interactions ◽

Zika Virus ◽

Coiled Coil ◽

Type I ◽

Protein Protein Interactions ◽

Pathogenic Potential ◽

Coiled Coil Domain ◽

Zika Virus Infection ◽

Type Iii Ifn

The ability of viruses to evade the host antiviral immune system determines their level of replication fitness, species specificity, and pathogenic potential. Flaviviruses rely on the subversion of innate immune barriers including the type I and type III IFN antiviral systems. Zika virus infection induces the degradation of STAT2, an essential component of the IFN stimulated gene transcription factor, ISGF3. The mechanisms that lead to STAT2 degradation by Zika virus are poorly understood, but it is known to be mediated by the viral NS5 protein that binds to STAT2 and targets it for proteasome-mediated destruction. To better understand how NS5 engages and degrades STAT2, functional analysis of the protein interactions that lead to Zika virus and NS5-dependent STAT2 proteolysis were investigated. Data implicate the STAT2 coiled-coil domain as necessary and sufficient for NS5 interaction and proteasome degradation after Zika virus infection. Molecular dissection reveals that the first two α-helices of the STAT2 coiled-coil contain a specific targeting region for IFN antagonism. These functional interactions provide a more complete understanding of the essential protein-protein interactions needed for Zika virus evasion of the host antiviral response, and identifies new targets for antiviral therapeutic approaches.

Download Full-text

Coiled-coil networking shapes cell molecular machinery

Molecular Biology of the Cell ◽

10.1091/mbc.e12-05-0396 ◽

2012 ◽

Vol 23 (19) ◽

pp. 3911-3922 ◽

Cited By ~ 44

Author(s):

Yongqiang Wang ◽

Xinlei Zhang ◽

Hong Zhang ◽

Yi Lu ◽

Haolong Huang ◽

...

Keyword(s):

Protein Interactions ◽

Critical Role ◽

Coiled Coil ◽

Coiled Coils ◽

Protein Protein Interactions ◽

Full Spectrum ◽

Kinetochore Assembly ◽

Genome Wide ◽

Molecular Machinery ◽

Systematic Understanding

The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications.

Download Full-text

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m109.001743 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16369-16376 ◽

Cited By ~ 23

Author(s):

Xuebo Hu ◽

Sungkwon Kang ◽

Xiaoyue Chen ◽

Charles B. Shoemaker ◽

Moonsoo M. Jin

Keyword(s):

Protein Interactions ◽

Secretory Pathway ◽

Coiled Coil ◽

Affinity Maturation ◽

Antibody Binding ◽

Soluble Form ◽

Protein Protein Interactions ◽

Yeast Surface ◽

Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

Download Full-text

Saccharomyces cerevisiae as a Tool to Investigate Plant Potassium and Sodium Transporters

International Journal of Molecular Sciences ◽

10.3390/ijms20092133 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2133 ◽

Cited By ~ 4

Author(s):

Antonella Locascio ◽

Nuria Andrés-Colás ◽

José Miguel Mulet ◽

Lynne Yenush

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Interactions ◽

Model System ◽

Protein Protein Interactions ◽

Alkali Cations ◽

Yeast Saccharomyces Cerevisiae ◽

Future Directions ◽

Sodium Transporters ◽

Adverse Environmental Conditions ◽

Sodium And Potassium

Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker’s yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein–protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.

Download Full-text

Divisome under Construction: Distinct Domains of the Small Membrane Protein FtsB Are Necessary for Interaction with Multiple Cell Division Proteins

Journal of Bacteriology ◽

10.1128/jb.01597-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2815-2825 ◽

Cited By ~ 40

Author(s):

Mark D. Gonzalez ◽

Jon Beckwith

Keyword(s):

Cell Division ◽

Membrane Proteins ◽

Protein Interactions ◽

Cell Envelope ◽

Coiled Coil ◽

Main Function ◽

Protein Protein Interactions ◽

Molecular Scaffold ◽

C Terminus ◽

Under Construction

ABSTRACT Cell division in bacteria requires the coordinated action of a set of proteins, the divisome, for proper constriction of the cell envelope. Multiple protein-protein interactions are required for assembly of a stable divisome. Within the Escherichia coli divisome is a conserved subcomplex of inner membrane proteins, the FtsB/FtsL/FtsQ complex, which is necessary for linking the upstream division proteins, which are predominantly cytoplasmic, with the downstream division proteins, which are predominantly periplasmic. FtsB and FtsL are small bitopic membrane proteins with predicted coiled-coil motifs, which themselves form a stable subcomplex that can recruit downstream division proteins independently of FtsQ; however, the details of how FtsB and FtsL interact together and with other proteins remain to be characterized. Despite the small size of FtsB, we identified separate interaction domains of FtsB that are required for interaction with FtsL and FtsQ. The N-terminal half of FtsB is necessary for interaction with FtsL and sufficient, when in complex with FtsL, for recruitment of downstream division proteins, while a portion of the FtsB C terminus is necessary for interaction with FtsQ. These properties of FtsB support the proposal that its main function is as part of a molecular scaffold to allow for proper formation of the divisome.

Download Full-text