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

• Type of Material: Article
• Authors: M Sherekar, National Laboratory for Cancer Research; Sae-Won Han, University of California San Francisco; Rodolfo Ghirlando, National Institutes of Health, Bethesda; Simon Messing, Frederick National Laboratory for Cancer Research; Matthew Drew, Frederick National Laboratory for Cancer Research; Dana Rabara, Frederick National Laboratory for Cancer Research; Timothy Waybright, Frederick National Laboratory for Cancer Research; Puneet Juneja, Emory University; Hugh O'Neill, Oak Ridge National Laboratory; Christopher B Stanley, Oak Ridge National Laboratory; Debsindhu Bhowmik, Oak Ridge National Laboratory; Arvind Ramanathan, Argonne National Laboratory; Sriram Subramaniam, Frederick National Laboratory for Cancer Research; Dwight V Nissley, Frederick National Laboratory for Cancer Research; William Gillette, Frederick National Laboratory for Cancer Research; Frank McCormick, Frederick National Laboratory for Cancer Research; Dominic Esposito, Frederick National Laboratory for Cancer Research
• Language: English
• Date: 2020-01-24
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2020 Sherekar et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.RA119.010934
• Title of Journal or Parent Work: JOURNAL OF BIOLOGICAL CHEMISTRY
• Volume: 295
• Issue: 4
• Start Page: 1105
• End Page: 1119
• Grant/Funding Information: This work was supported in whole or in part by National Institutes of Health under NCI Contract HHSN261200800001E and the Intramural Research Program of the NIDDK. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health
• Keywords: Ras protein; dimerization; GTPase Kras (KRAS); GTPase-activating protein (GAP); NF1; cell signaling; mitogen-activated protein kinase pathway; cancer; neurofibromin
• Research Categories: Chemistry, Biochemistry; Health Sciences, Medicine and Surgery; Biology, Molecular; Health Sciences, Oncology

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.