The structure of neurofibromin isoform 2 reveals different functional states

Abstract

The autosomal dominant monogenetic disease neurofibromatosis type 1 (NF1) affects approximately one in 3,000 individuals and is caused by mutations in the NF1 tumour suppressor gene, leading to dysfunction in the protein neurofibromin (Nf1)1,2. As a GTPase-activating protein, a key function of Nf1 is repression of the Ras oncogene signalling cascade. We determined the human Nf1 dimer structure at an overall resolution of 3.3 Å. The cryo-electron microscopy structure reveals domain organization and structural details of the Nf1 exon 23a splicing3 isoform 2 in a closed, self-inhibited, Zn-stabilized state and an open state. In the closed conformation, HEAT/ARM core domains shield the GTPase-activating protein-related domain (GRD) so that Ras binding is sterically inhibited. In a distinctly different, open conformation of one protomer, a large-scale movement of the GRD occurs, which is necessary to access Ras, whereas Sec14-PH reorients to allow interaction with the cellular membrane4. Zn incubation of Nf1 leads to reduced Ras-GAP activity with both protomers in the self-inhibited, closed conformation stabilized by a Zn binding site between the N-HEAT/ARM domain and the GRD–Sec14-PH linker. The transition between closed, self-inhibited states of Nf1 and open states provides guidance for targeted studies deciphering the complex molecular mechanism behind the widespread neurofibromatosis syndrome and Nf1 dysfunction in carcinogenesis.

Structural basis of activation of the tumor suppressor protein neurofibromin

“…While individual domains of neurofibromin have been crystalized (D'angelo et al, 2006;Scheffzek et al, 1998), the overall architecture of the protein has long remained elusive. Recently, structures of neurofibromin solved by cryoelectron microscopy (cryo-EM) have revealed the dimeric architecture of this protein, which rests in an equilibrium between two conformational states (Lupton et al, 2021;Naschberger et al, 2021), including an autoinhibited closed conformation. However, the mechanism of neurofibromin activation remains unclear, impeded by lower resolution of the open-active conformation.…”

RAS and beyond: the many faces of the neurofibromatosis type 1 protein

“…Given that neurofibromin is a large molecule with numerous protein-binding domains and novel conformational states ( Naschberger et al, 2021 ), it is likely that other, perhaps RAS-independent, properties exist.…”

“…Although there is a wealth of information delineating the varied functions of neurofibromin in different cell types and tissues, most of these studies are focused on RAS pathway regulation. The recent elucidation of the physical structure neurofibromin revealed that it likely functions as a homodimer ( Lupton et al, 2021 ; Naschberger et al, 2021 ; Sherekar et al, 2020 ) with at least two distinct molecular conformations. In its closed, auto-inhibited conformation, RAS binding is blocked, thus inhibiting neurofibromin RAS-GAP activity.…”

“…In its closed, auto-inhibited conformation, RAS binding is blocked, thus inhibiting neurofibromin RAS-GAP activity. However, in its open conformation, the lipid-binding SEC-PH domain of one neurofibromin protomer interacts with the cell membrane to expose and activate the RAS-GAP domain ( Naschberger et al, 2021 ). Neurofibromin forms dimers to bind and hydrolyze RAS molecules, and neurofibromin monomers and dimers are similarly efficient in their RAS-GAP activity in vitro ( Sherekar et al, 2020 ).…”

“…Given the diverse number of clinical manifestations involving many distinct cell types (https:// www.proteinatlas.org/ENSG00000196712-NF1/tissue), as well as the growing number of potential neurofibromin-interacting proteins (Fig. 2A), which will likely expand following the recent crystallization of the neurofibromin dimer protein (Lupton et al, 2021;Naschberger et al, 2021), it is conceivable that neurofibromin may have functions beyond simple canonical RAS pathway regulation. In this Special article, we will detail what is known about NF1 protein function and discuss evidence supporting both RAS-dependent and RAS-independent mechanisms for neurofibromin regulation of cell biology.…”