Designed membrane protein heterodimers and control of their affinity by binding domain and membrane linker properties.

Chenyang Lan,Heiko Heerklotz,Manfred Jung,Anja Stulz,Susan Lauw,Nicolas P F Barthes,Pavel Salavei,Maximilian H Ulbrich

doi:10.1039/d1nr06574b

Abstract

Many membrane proteins utilize dimerization to transmit signals across the cell membrane via regulation of the lateral binding affinity. The complexity of natural membrane proteins hampers the understanding of this regulation on a biophysical level. We designed simplified membrane proteins from well-defined soluble dimerization domains with tunable affinities, flexible linkers, and an inert membrane anchor. Live-cell single-molecule imaging demonstrates that their dimerization affinity indeed depends on the strength of their binding domains. We confirm that as predicted, the 2-dimensional affinity increases with the 3-dimensional binding affinity of the binding domains and decreases with linker lengths. Models of extended and coiled linkers delineate an expected range of 2-dimensional affinities, and our observations for proteins with medium binding strength agree well with the models. Our work helps in understanding the function of membrane proteins and has important implications for the design of synthetic receptors.

Highlights

Most membrane proteins laterally interact with other membrane proteins
In contrast to ion channels that form rigid multi-subunit structures spanning the extracellular, transmembrane, and intracellular regions, proteins that use a shift in the monomer/dimer equilibrium as a tool to transduce signals across the membrane, e.g. receptor tyrosine kinases, often have interaction domains that are well separated by flexible connections
We chose a modular design with exchangeable components that carry the functionalities of dimerization domains, membrane anchors, and fluorescent markers for microscopy-based readout

Summary

Introduction

In contrast to ion channels that form rigid multi-subunit structures spanning the extracellular, transmembrane, and intracellular regions, proteins that use a shift in the monomer/dimer equilibrium as a tool to transduce signals across the membrane, e.g. receptor tyrosine kinases, often have interaction domains that are well separated by flexible connections. In this way, the propensity to form dimers can be regulated by soluble extra- or intracellular ligands, or a change in the membrane composition.[1,2] Effectively, the regulation is a change of the molecules’ 2-dimensional affinity for each other. With a low density of the proteins in the membrane, heterodimers appeared as yellow spots, whereas monomers were green and red spots

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Results

Conclusion