The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS

• Published in: Structure (London) 2015-08, Vol.23 (8), p.1382-1393
• Main Authors: Aramini, James M., Vorobiev, Sergey M., Tuberty, Lynda M., Janjua, Haleema, Campbell, Elliot T., Seetharaman, Jayaraman, Su, Min, Huang, Yuanpeng J., Acton, Thomas B., Xiao, Rong, Tong, Liang, Montelione, Gaetano T.
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Amino Acid Sequence; Binding Sites; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Humans; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Proto-Oncogene Proteins B-raf - chemistry; Proto-Oncogene Proteins B-raf - genetics; Proto-Oncogene Proteins p21(ras) - chemistry; Proto-Oncogene Proteins p21(ras) - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Sequence Alignment; Signal Transduction
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2015.06.003

RAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and 19F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase. [Display omitted] •Solution NMR and X-ray crystal structures of BRAF RAS-binding domain•RAS-binding surface mapped onto the structure of BRAF RBD•RAS binding alters amide exchange rates throughout BRAF RBD•Evidence for RAS-induced allosteric changes in BRAF RBD RAS binding is a critical step in the activation of BRAF kinase and the MAPK signaling pathway. Aramini et al. report the solution NMR and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF, and use NMR to reveal unexpected allosteric changes in BRAF RBD upon RAS binding.