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

Abstract

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase 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–multiangle 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 EM 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 co-expression 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.

Neurofibromin Structure, Functions and Regulation

“…This dimerization was shown to take place in vivo in human cells and was visualized as a pseudo-symmetric dimeric particle after transmission electron microscopy analysis of projection images of negatively stained neurofibromin. Domains mediating neurofibromin dimerization were shown to be the TBD (tubulinbinding domain), which consists of three predicted amphipathic alpha-helices and a region localized between PH and CTD domains that contains a series of 12 predicted HEAT-like repeats commonly involved in protein-protein interactions, and often forming a complex solenoid structure incorporating a series of alpha-helices [43]. The authors emphasize that this kind of interaction has already been described with high affinity mTOR dimers.…”

“…To date, only the 3D structures of the GRD and SecPH domains have been resolved, representing 25% of the entire protein. However, recently, structural data have been obtained on full-length neurofibromin using a series of biochemical and biophysical experiments indicating that full-length neurofibromin forms a high-affinity dimer [43].…”

“…Size-exclusion chromatography multi-angle light scattering (SEC-MALS), small-angle X-ray and neutron scattering (SAXS and SANS, respectively), and analytical ultracentrifugation performed on purified full-length human neurofibromin produced in baculovirus-infected insect cells, demonstrated that full-length neurofibromin forms a high-affinity dimer [43]. This dimerization was shown to take place in vivo in human cells and was visualized as a pseudo-symmetric dimeric particle after transmission electron microscopy analysis of projection images of negatively stained neurofibromin.…”

“…Carnes et al (2019) [147] were the first to show neurofibromin oligomerisation by studying neurofibromin interactome after affinity purification. Recently, Sherekar et al (2020) [43] further showed that neurofibromin forms high-affinity dimers in vitro and in cells. Regions responsible for neurofibromin dimerization were mapped between amino acids 1085 and 1172, containing the TBD, and between amino acids 1821 and 2018 (localized after the PH domain and before the CTD).…”

“…In addition, a mixture of the N-terminal and C-terminal fragments that harbor the dimerization regions can reconstitute full-length dimeric neurofibromin structures with similar high affinity. The biological consequences of neurofibromin dimerization are still unclear [43].…”