The cryo-EM structure of the neurofibromin dimer reveals the molecular basis for von Recklinghausen disease

Abstract

Neurofibromin (NF1) is a tumour suppressor mutated in neurofibromatosis type 1 (von Recklinghausen disease), one of the most common human genetic diseases. NF1 regulates cellular growth through suppressing the Rat Sarcoma (RAS) pathway and, accordingly, mutations in this protein drive numerous cancers, including melanoma, ovarian, breast and brain cancer. Currently, however, the molecular basis for NF1 function remains to be understood. Here we address this problem and use cryogenic Electron Microscopy (cryo-EM) to determine the structure of full-length NF1. The 640 kDa NF1 homodimer forms an extraordinary lemniscate (∞) shaped molecule that is ~30 nm in length and ~10 nm wide. Each NF1 monomer comprises an N-terminal HEAT-repeat domain (N-HEAT), a guanosine triphosphatase activating protein (GAP)-related domain (GRD), a Sec14 homologous and pleckstrin homologous module (SEC-PH), and a C-terminal HEAT domain (C-HEAT). The core NF1 scaffold is formed via a head-to-tail dimer of the N- and C-HEAT domains. This platform, which is responsible for interacting with more than 10 regulatory binding partners, comprises an extraordinary array of over 150 α-helices. Analysis of these EM data revealed that the GRD and SEC-PH domain are highly mobile with respect to the core scaffold and could not initially be accurately placed in electron density. Strikingly, however, using 3D variability analysis we were able to identify a significant subpopulation of NF1 particles and determine the complete NF1 structure to 5.6 Å resolution. These data revealed that the catalytic GRD and lipid binding SEC-PH domain are positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation in order to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Finally, it is suggested that approaches to combat NF1-linked diseases may include release of the autoinhibited state in order to improve NF1 catalytic efficiency.

Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins

“…Our results show that the direction of the ΔNLS or NLS knockdown effects is most often opposite and suggest that the two functionally interact when both present. Whether this occurs through the formation of a dimer, if neurofibromins form dimers [28,68] in eukaryotic cells at normal neurofibromin concentrations, is an intriguing question. Indeed, how the NLS and ΔNLS conformations may affect dimer formation is an interesting experimental goal.…”

Clinical and Basic Aspects of Neurofibromatosis Type 1

“…Our results show that the direction of the ΔNLS or NLS knockdown effects is most often opposite and suggest that the two functionally interact when both present. Whether this occurs through the formation of a dimer, if neurofibromins form dimers [28,68] in eukaryotic cells at normal neurofibromin concentrations, is an intriguing question. Indeed, how the NLS and ΔNLS conformations may affect dimer formation is an interesting experimental goal.…”

Neurofibromatosis Type 1 Gene Alterations Define Specific Features of a Subset of Glioblastomas

“… Domain architecture of neurofibromin: The lemniscate NF1 complex is formed by a head-to-tail dimer of an N-terminal HEAT domain (N-HEAT) and a C-terminal HEAT domain (C-HEAT) [ 58 ]. The GRD domain possesses a Ras-GAP function.…”

“…It is involved in membrane localisation through the binding with lipids, actin remodelling through the Rho–ROCK pathway, and dendritic spine formation through VCP. As a neurofibromin creates a high-affinity dimer, on the bottom with the gray colour are shown primary dimerisation interfaces [ 58 ]. The figure was created with BioRender.com (XR2390NUCR, 27.11.2021) and adapted from [ 68 , 69 ].…”