High-throughput discovery of transmembrane helix dimers from human single-pass membrane proteins with TOXGREEN sort-seq

The oligomerization of the transmembrane helices of single-pass membrane
proteins is crucial to biological function, and its misregulation can lead
to many diseases. The study of transmembrane helix oligomerization is
facilitated by the availability of genetic reporter assays, which are
essential tools for understanding the organization and biology of
single-pass systems. In particular, reporter assays are crucial for
mapping the oligomerization interfaces of transmembrane helices through
scanning mutagenesis, but their application is limited by the need to
clone and measure each construct individually. Here, we present “TOXGREEN
sort-seq”, a high-throughput version of the TOXGREEN assay that enables
the direct measurement of transmembrane helix oligomerization in large
libraries using fluorescence-activated cell sorting and next-generation
sequencing. We show that TOXGREEN sort-seq is robust and reproduce the
direct measurements of individual constructs with good accuracy and
sensitivity. The method produced high-quality mutational profiles from a
library of 17,400 constructs designed to probe the interface of 100
potential GASrightdimers predicted from sequences of human single-pass
membrane proteins. We report the validated structural model of twelve
dimers involved in a variety of biological functions, including immune
response (interleukin-22 receptor subunit alpha-1, butyrophilin-like
protein 3, hepatitis A virus cellular receptor 2), transport (transferrin
receptor protein 1), and cell-surface signaling and proliferation
(syndecan-3; semaphorins 5A, 6B and 6D). Remarkably, all three semaphorins
in the dataset formed strong dimers and produced mutational profiles
consistent with the computational structure. These findings open the
possibility that dimerization may be relevant to these proteins’ activity
and provide a validated interface for assessing their biological role.