Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Neurofibromin is a tumor suppressor encoded by the NF1 gene which is mutated in the Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase (MAPK) pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein-to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments including size-exclusion chromatography-multi angle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications.  Analysis of purified full-length and truncated neurofibromin variants by negative- stain electron microscopy revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and coexpression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together these results suggest how neurofibromin dimers might form and be stabilized within the cell. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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