Cadherin clusters stabilized by a combination of specific and nonspecific cis-interactions.

Connor J Thompson,Deborah E Leckband,Vinh H Vu,Daniel K Schwartz,Zhaoqian Su,Yinghao Wu

doi:10.7554/elife.59035

Abstract

We demonstrate a combined experimental and computational approach for the quantitative characterization of lateral interactions between membrane-associated proteins. In particular, weak, lateral (cis) interactions between E-cadherin extracellular domains tethered to supported lipid bilayers, were studied using a combination of dynamic single-molecule Förster Resonance Energy Transfer (FRET) and kinetic Monte Carlo (kMC) simulations. Cadherins are intercellular adhesion proteins that assemble into clusters at cell-cell contacts through cis- and trans- (adhesive) interactions. A detailed and quantitative understanding of cis-clustering has been hindered by a lack of experimental approaches capable of detecting and quantifying lateral interactions between proteins on membranes. Here single-molecule intermolecular FRET measurements of wild-type E-cadherin and cis-interaction mutants combined with simulations demonstrate that both nonspecific and specific cis-interactions contribute to lateral clustering on lipid bilayers. Moreover, the intermolecular binding and dissociation rate constants are quantitatively and independently determined, demonstrating an approach that is generalizable for other interacting proteins.

Highlights

The quantitative characterization of protein interactions on membranes and at buried interfaces, including the measurement of binding constants, is a major challenge due to the limited experimental approaches capable of interrogating molecular interactions in these environments
The diffusion coefficient served as a very sensitive proxy for cis-interactions, 79 because clusters diffuse more slowly than monomers. These findings suggested that cis-interactions 80 between E-cad extracellular domains can result in the formation of large clusters, in the absence of trans81 interactions, for cadherin surface coverage above a threshold of ~1,100 E-cad/μm2 (Thompson et al, 2019)
The single molecule Förster Resonance Energy Transfer (FRET) measurements revealed that both specific and nonspecific cis-interactions contribute to wild-type E-cadherin clustering at a physiologically relevant surface coverage

Summary

Introduction

The quantitative characterization of protein interactions on membranes and at buried interfaces, including the measurement of binding constants, is a major challenge due to the limited experimental approaches capable of interrogating molecular interactions in these environments. The immunological synapse is characterized by the spatial and temporal organization of proteins in the gaps between the surface of an antigen presenting cell and a T-cell (Grakoui et al, 1999; Monks, Freiberg, Kupfer, Sciaky, & Kupfer, 1998). This organization is attributed in part to the steric segregation of proteins of different sizes and to cytoskeletal interactions (Qi, Groves, & Chakraborty, 2001; Schmid et al, 2016); the understanding of the role of lateral protein interactions in this protein assembly remains incomplete. It is possible to quantify trans- (adhesive) interactions (Chesla, Selvaraj, & Zhu, 1998; Chien et al., 2008; J. Wu et al, 2008), measurements of lateral interactions underlying protein clustering have been inaccessible

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