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bioRxiv · 10.1101/2025.04.22.650048

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

Anderson, S. M.·Choi, J.·Cushman, E. M.·Leander, M.·Raman, S.·Senes, A.
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Abstract

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 GASright dimers 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.

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Anderson, S. M., Choi, J., Cushman, E. M., Leander, M., Raman, S., Senes, A.. 2025-04-26. High-throughput discovery of transmembrane helix dimers from human single-pass membrane proteins with TOXGREEN sort-seq. https://doi.org/10.1101/2025.04.22.650048

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