A two-hybrid antibody micropattern assay reveals specific in cis interactions of MHC I heavy chains at the cell surface.

Cindy Dirscherl,Sebastian Springer,Catherine Jacob-Dolan,Venkat Raman Ramnarayan,Zeynep Hein

doi:10.7554/elife.34150

Abstract

We demonstrate a two-hybrid assay based on antibody micropatterns to study protein-protein interactions at the cell surface of major histocompatibility complex class I (MHC I) proteins. Anti-tag and conformation-specific antibodies are used for individual capture of specific forms of MHC I proteins that allow for location- and conformation-specific analysis by fluorescence microscopy. The assay is used to study the in cis interactions of MHC I proteins at the cell surface under controlled conditions and to define the involved protein conformations. Our results show that homotypic in cis interactions occur exclusively between MHC I free heavy chains, and we identify the dissociation of the light chain from the MHC I protein complex as a condition for MHC I in cis interactions. The functional role of these MHC I protein-protein interactions at the cell surface needs further investigation. We propose future technical developments of our two-hybrid assay for further analysis of MHC I protein-protein interactions.

Highlights

Protein-protein interactions are difficult to investigate, especially when they involve membrane proteins under physiological conditions, specific protein conformations or subpopulations, low affinities, or defined locations in the cell
We demonstrate an expansion of this concept to characterize locationand conformation-specific protein-protein interactions in a novel two-hybrid assay read out by fluorescence microscopy by using microprinted antibody patterns for the capture of bait proteins to spatially arrange them in the plasma membrane of live cells (Dirscherl and Springer, 2017) and to investigate their interaction with green fluorescent protein (GFP)-tagged prey proteins
We first tested whether the two common b2m-dependent monoclonal antibodies Y3 and 27-11-13S were still specific for their target allotypes when used in the pattern capture assay

Summary

Introduction

Protein-protein interactions are difficult to investigate, especially when they involve membrane proteins under physiological conditions, specific protein conformations or subpopulations, low affinities, or defined locations in the cell. Such challenges are not usually met by yeast two-hybrid screens and co-immunoprecipitation approaches; instead, they require technically demanding methods such as fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy, which only work in some cases. Our work shows conclusively that MHC I free heavy chains, but not HC/b2m/peptide trimers or HC/b2m dimers, associate in cis at the plasma membrane

Results

Discussion

Materials and methods

Funding Funder