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

Mukul Sherekar, Sae Won Han, Rodolfo Ghirlando, Simon Messing, Matthew Drew, Dana Rabara, Timothy Waybright, Puneet Juneja, Hugh O'Neill, Christopher B. Stanley, Debsindhu Bhowmik, Arvind Ramanathan, Sriram Subramaniam, Dwight V. Nissley, William Gillette, Frank McCormick, Dominic Esposito

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

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 fulllength 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 fulllength 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.

Original languageEnglish
Pages (from-to)1105-1119
Number of pages15
JournalJournal of Biological Chemistry
Volume295
Issue number4
DOIs
StatePublished - Jan 24 2020

Funding

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 Ser-vices nor does mention of trade names, commercial products, or organiza-tions 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. 2 Supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory for SAS and EM characterization.

FundersFunder number
Oak Ridge National Laboratory
National Institutes of Health
National Cancer InstituteHHSN261200800001E
National Institute of Diabetes and Digestive and Kidney DiseasesZIADK033007

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