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

Abstract

SUMMARY 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.

Water‐Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase

“…[27,28] In this study, by contrast, three observations suggest that mutations do not bring The mechanisms by which mutations reorganize water are apparent in the WaterMappredicted hydration sites near the amino acid substitution for which H/S compensation was most pronounced: L198A (Fig. 2C).…”

Raf-1 Cysteine-Rich Domain Increases the Affinity of K-Ras/Raf at the Membrane, Promoting MAPK Signaling

“…Ras interacts with Raf RBD through the effector binding site with high affinity (Chuang et al, 1994;Herrmann et al, 1995). Several structural studies targeted the Ras/Raf-RBD complex exploiting the RBD crystal structures in complex with Ras (Aramini et al, 2015;Fetics et al, 2015;Kauke et al, 2017;Walker et al, 2014;Wan et al, 2004;Zeng et al, 1999). NMR and X-ray crystal structure studies illustrated that upon Ras binding, B-Raf RBD undergoes conformational change with the allosteric signal propagating from the GppNHp-bound H-Ras to the core region of the RBD (Aramini et al, 2015).…”

“…Several structural studies targeted the Ras/Raf-RBD complex exploiting the RBD crystal structures in complex with Ras (Aramini et al, 2015;Fetics et al, 2015;Kauke et al, 2017;Walker et al, 2014;Wan et al, 2004;Zeng et al, 1999). NMR and X-ray crystal structure studies illustrated that upon Ras binding, B-Raf RBD undergoes conformational change with the allosteric signal propagating from the GppNHp-bound H-Ras to the core region of the RBD (Aramini et al, 2015). For Raf-1 RBD in complex with the GppNHp-bound H-Ras, X-ray crystal studies combined with molecular dynamics (MD) simulations demonstrated that allosteric signals propagate through pathways connecting the Ras allosteric lobe to Raf-1 RBD (Fetics et al, 2015).…”

A “Tug of War” maintains a dynamic protein-membrane complex as shown in all-atom simulations of C-Raf RBD-CRD bound to K-Ras4B at an anionic membrane

“…Overall, these detailed protein-protein contacts are in close agreement with the interactions seen in the experimental structure (Fetics et al, 2015). Two reports also suggested a direct interaction between the CRD and the switch II region of Ras.GTP (Aramini et al, 2015;Brtva et al, 1995). These interactions are not observed, however, in any of the 5 simulations ( Fig.…”

“…In the present investigation we studied the conformational and orientational dynamics of the C-Raf RBD-CRD : K-Ras complex bound to a membrane model. That the Ras Binding Domain, or , makes direct contacts with the membrane-bound Ras is well established and structures for binding of the K-Ras4B homologous H-Ras GTPase to Raf have been presented (Aramini et al, 2015;Fetics et al, 2015). There is as yet no study that clearly shows a direct interaction between Ras and the Cysteine Rich Domain, which follows the RBD in the sequence Raf, although a number of studies have indicated such interactions (Brtva et al, 1995;Clark et al, 1996).…”