Allosteric modulation of NF1 GAP: Differential distributions of catalytically competent populations in loss-of-function and gain-of-function mutants

Neurofibromin (NF1), a Ras GTPase-activating protein (GAP), catalyzes Ras-mediated GTP hydrolysis and thereby negatively regulates the Ras/MAPK pathway. NF1 mutations can cause neurofibromatosis type 1 manifesting tumors, and neurodevelopmental disorders. Exactly how the missense mutations in the GAP-related domain of NF1 (NF1GRD) allosterically impact NF1 GAP to promote these distinct pathologies is unclear. Especially tantalizing is the question of how same-domain, same-residue NF1GRD variants exhibit distinct clinical phenotypes. Guided by clinical data, we take up this dilemma. We sampled the conformational ensembles of NF1GRD in complex with GTP-bound K-Ras4B by performing molecular dynamics simulations. Our results show that mutations in NF1GRD retain the active conformation of K-Ras4B but with biased propensities of the catalytically competent populations of K-Ras4B-NF1GRD complex. In agreement with clinical depiction and experimental tagging, compared to the wild type, NF1GRD E1356A and E1356V mutants effectively act through loss-of-function and gain-of-function mechanisms, leading to neurofibromatosis and developmental disorders, respectively. Allosteric modulation of NF1GRD GAP activity through biasing the conformational ensembles in the different states is further demonstrated by the diminished GAP activity by NF1GRD isoform 2, further manifesting propensities of conformational ensembles as powerful predictors of protein function. Taken together, our work identifies a NF1GRD hotspot that could allosterically tune GAP function, suggests targeting Ras oncogenic mutations by restoring NF1 catalytic activity, and offers a molecular mechanism for NF1 phenotypes determined by their distinct conformational propensities. © 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.

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