scholarly journals Mapping transmembrane binding partners for E-cadherin ectodomains

2020 ◽  
Vol 117 (49) ◽  
pp. 31157-31165
Author(s):  
Omer Shafraz ◽  
Bin Xie ◽  
Soichiro Yamada ◽  
Sanjeevi Sivasankar
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Binding Assays ◽  
Adhesion Protein ◽  
Bait Protein ◽  
Binding Partners ◽  
Extracellular Region ◽  
Focal Adhesion Protein ◽  
E Cadherin

We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a “bait” protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell–cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2β1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.

scholarly journals Mapping transmembrane binding partners for E-cadherin ectodomains

2020 ◽  
Author(s):  
Omer Shafraz ◽  
Bin Xie ◽  
Soichiro Yamada ◽  
Sanjeevi Sivasankar
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Binding Assays ◽  
Binding Interactions ◽  
Adhesion Protein ◽  
Bait Protein ◽  
Binding Partners ◽  
Extracellular Region ◽  
E Cadherin

ABSTRACTWe combine proximity labeling and single molecule binding assays, to discover novel transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a bait protein with TurboID biotin ligase, and identify proximal proteins that are biotin-tagged on both their extracellular and intracellular regions. We then test direct binding interactions between the proximal proteins and the bait, using single molecule Atomic Force Microscope binding assays. Using this approach, we identify novel binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2β1, and the receptor tyrosine kinase ligand ephrin-B1, all directly interact with E-cadherin ectodomains. Our discovery of previously unknown heterophilic E-cadherin binding interactions, suggest the existence of novel cadherin cross-talk in epithelial cells.

scholarly journals Diffusion of the disordered E-cadherin tail on β-catenin

2021 ◽  
Author(s):  
Felix Wiggers ◽  
Samuel Wohl ◽  
Artem Dubovetskyi ◽  
Gabriel Rosenblum ◽  
Wenwei Zheng ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Single Molecule ◽  
Protein Interactions ◽  
Energy Landscape ◽  
Specific Protein ◽  
Adhesion Protein ◽  
High Affinity ◽  
The Stability ◽  
Binding Interfaces ◽  
E Cadherin

AbstractSpecific protein interactions typically require well-shaped binding interfaces. Here, we report a cunning exception. The disordered tail of the cell-adhesion protein E-cadherin dynamically samples a large surface area of the proto-oncogene β-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without dissociation of the complex. A few persistent interactions provide specificity whereas unspecific contacts boost affinity. The energy landscape of this complex is rugged with many small barriers (3 – 4 kBT) and reconciles specificity, high affinity, and extreme disorder. Given the roles of β-catenin in cell-adhesion, signalling, and cancer, this Velcro-like design has the potential to tune the stability of the complex without requiring dissociation.

scholarly journals Spatial and temporal organization of cadherin in punctate adherens junctions

2018 ◽  
Vol 115 (19) ◽  
pp. E4406-E4415 ◽  
Author(s):  
Indrajyoti Indra ◽  
Jongho Choi ◽  
Chi-Shuo Chen ◽  
Regina B. Troyanovsky ◽  
Lawrence Shapiro ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transmembrane Protein ◽  
Adherens Junctions ◽  
Temporal Organization ◽  
A431 Cells ◽  
Alternative Strategies ◽  
Localization Microscopy ◽  
Tissue Integrity ◽  
Single Molecule Localization Microscopy ◽  
E Cadherin

Adherens junctions (AJs) play a fundamental role in tissue integrity; however, the organization and dynamics of the key AJ transmembrane protein, E-cadherin, both inside and outside of AJs, remain controversial. Here we have studied the distribution and motility of E-cadherin in punctate AJs (pAJs) of A431 cells. Using single-molecule localization microscopy, we show that pAJs in these cells reach more than 1 μm in length and consist of several cadherin clusters with crystal-like density interspersed within sparser cadherin regions. Notably, extrajunctional cadherin appears to be monomeric, and its density is almost four orders of magnitude less than observed in the pAJ regions. Two alternative strategies of tracking cadherin motion within individual junctions show that pAJs undergo actin-dependent rapid—on the order of seconds—internal reorganizations, during which dense clusters disassemble and their cadherins are immediately reused for new clusters. Our results thus modify the classical view of AJs by depicting them as mosaics of cadherin clusters, the short lifetimes of which enable stable overall morphology combined with rapid internal rearrangements.

scholarly journals Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nitesh Shashikanth ◽  
Meridith A. Kisting ◽  
Deborah E. Leckband
Keyword(s):  
Protein Interactions ◽  
Intercellular Junctions ◽  
Binding Kinetics ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Kinetic Measurements ◽  
Adhesion Protein ◽  
Membrane Bound ◽  
And Function ◽  
The Impact

Abstract The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions.

Identification of binding partners of CsaA - an archaeal chaperonic pro-tein of Picrophilus torridus

2020 ◽  
Vol 27 ◽  
Author(s):  
Neelja Singhal ◽  
Archana Sharma ◽  
Manisha Aswal ◽  
Nirpendra Singh ◽  
Manish Kumar ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Citrate Synthase ◽  
Elongation Factor ◽  
Trna Synthetase ◽  
Strong Interactions ◽  
Beta Chain ◽  
Protein Protein Interactions ◽  
Uncharacterized Protein ◽  
Binding Partners ◽  
Picrophilus Torridus

Background:: CsaA is among the few chaperones which are present in both bacteria and archaea, but absent in eukaryotes. There are no reports on interactome analysis of CsaA from archaea, till date. Identification of binding partners of CsaA might be helpful in understanding CsaA-associated processes in Picrophilus torridus– an extreme thermoaci-dophilic euryarchaeon. Objectives:: The present study was conducted to identify the binding partners of CsaA of P. torridus (PtCsaA). Methods:: The binding partners of PtCsaA were isolated and identified using a pull down assay and liquid chromatography-mass spectrometry (LC-MS). Results:: The results revealed twelve potential binding partners of CsaA. These were thermosome subunits (Q6KZS2 and Q6L132), nascent polypeptide-associated complex protein (Q6L1N3), elongation factor 1-alpha (Q6L202), uncharacterized protein (Q6L0Y6), citrate synthase (Q6L0M8), asparaginyl-tRNA synthetase (Q6L0M5), succinyl-CoA synthetase beta chain (Q6L0B4), pyruvate ferredoxin oxidoreductase alpha and beta chain proteins (Q6KZA7 and Q6KZA6, respectively), malate dehydrogenase (Q6L0C3) and reversed fumarylacetoacetase (Q6KZ97). Functional categorization revealed that of these, six proteins were involved in energy metabolic pathways, three were archaeal chaperones, two were involved in trans-lation and one might be a transcription regulator. STRING-based analysis of the protein-protein interactions of the experi-mental interactome revealed strong interactions among them. Conclusion:: PtCsaA might be a multifaceted protein which besides translation might also play important role in metabolic processes of P. torridus. However, further experiments investigating the binding partners of CsaA in other archaea are re-quired for a better understanding of CsaA-associated processes in archaea.

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