NF2 (Neurofibromine 2, merlin) is frequently inactivated in cancer, where its NF2 tumor suppressor functionality is tightly coupled to protein conformation. How NF2 conformation is regulated and how NF2 conformation influences tumor suppressor activity is a largely open question. Here we systematically characterized three NF2 conformation-dependent protein interactions utilizing deep mutational scanning interaction perturbation analyses. We identified two regions in NF2 with clustered mutations which affected conformation dependent protein interactions. NF2 variants in the F3 subdomain and the alpha 3H helix region substantially modulated NF2 conformation and homomerization. Mutations in the F3 subdomain altered proliferation in three cell lines and matched patterns of disease mutations in neurofibromatosis. This study highlights the power of systematic mutational interaction perturbation analysis to identify missense variants impacting NF2 conformation and provides insight into NF2 tumor suppressor function.

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NF2 (moesin–ezrin–radixin-like [MERLIN] tumor suppressor) is frequently inactivated in cancer, where its NF2 tumor suppressor functionality is tightly coupled to protein conformation. How NF2 conformation is regulated and how NF2 conformation influences tumor suppressor activity is a largely open question. Here, we systematically characterized three NF2 conformation-dependent protein interactions utilizing deep mutational scanning interaction perturbation analyses. We identified two regions in NF2 with clustered mutations which affected conformation-dependent protein interactions. NF2 variants in the F2–F3 subdomain and the α3H helix region substantially modulated NF2 conformation and homomerization. Mutations in the F2–F3 subdomain altered proliferation in three cell lines and matched patterns of disease mutations in NF2 related-schwannomatosis. This study highlights the power of systematic mutational interaction perturbation analysis to identify missense variants impacting NF2 conformation and provides insight into NF2 tumor suppressor function.