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 (NF1^GRD) allosterically impact NF1 GAP to promote these distinct pathologies is unclear. Especially tantalizing is the question of how same-domain, same-residue NF1^GRD variants exhibit distinct clinical phenotypes. Guided by clinical data, we take up this dilemma. We sampled the conformational ensembles of NF1^GRD in complex with GTP-bound K-Ras4B by performing molecular dynamics simulations. Our results show that mutations in NF1^GRD retain the active conformation of K-Ras4B but with biased propensities of the catalytically competent populations of K-Ras4B-NF1^GRD complex. In agreement with clinical depiction and experimental tagging, compared to the wild type, NF1^GRD 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 NF1^GRD GAP activity through biasing the conformational ensembles in the different states is further demonstrated by the diminished GAP activity by NF1^GRD isoform 2, further manifesting propensities of conformational ensembles as powerful predictors of protein function. Taken together, our work identifies a NF1^GRD 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.