Fig 1.
Domain organization of RAS effectors and different proteins used in this study.
(A) Various domains are highlighted, including RAS association domain (RA) and RAS-binding (RB) domain in blue. The numbers indicate the N- and C-terminal amino acids of the respective effector domain used in this study. Other domains are: C1, cysteine-rich lipid binding; C2, calcium-dependent lipid binding; CRD, cysteine rich domains; DEP, Dishevelled/Egl-10/Pleckstrin; EF, EF-hand; kinase, serine/threonine or phosphoinositide kinase; PH, pleckstrin homology; PI3K, Phosphoinositide 3-kinase family, accessory domain; PP, proline-rich region; RA, RAS association; RALGEF, RAL specific guanine nucleotide exchange factor; RASGEF, RAS specific guanine nucleotide exchange factor; RB, RAS binding; REM, RAS exchanger motif; SARAH, Salvador/RASSF/Hippo. (B) Coomassie brilliant blue (CBB) stained SDS-PAGE of purified MBP fusion proteins used in this study.
Table 1.
Register of dissociation constants (Kd) determined for the RAS-effector interactions.
Fig 2.
Equilibrium dissociation constants for RAS-effector interaction determined Fluorescence polarization.
(A) Fluorescence polarization experiments were conducted by titrating mGppNHp-bound, active forms of RAS proteins (1 μM, respectively) with increasing concentrations of the respective effector domains as MBP fusion proteins. Data of two representative experiments for the interaction of KRAS (upper panel) and RRAS2 (lower panel) with CRAF-RB and PI3Kα-RB, respectively, are shown. All other data are illustrated in S1 Fig (B) Evaluated equilibrium dissociation constants (Kd) in μM shown as data points illustrate a significant difference in the binding properties of the effector proteins with both RAS and RRAS isoforms, respectively. A mean value of 0.94 ± 0.014 μM has been determined for the interaction between HRAS and CRAF to exemplify the reproducibility of this approach.
Table 2.
Dissociation constants (Kd) in μM for the interaction between RAS proteins and effectors.
Fig 3.
Superposition of all available RAS–effector complex structures.
Nine structures of RAS-effector domain complexes, found in a PDB search, including HRAS-CRAF (PDB code: 4g0n, 4g3x, 3kud; red), HRAS-BYR2 (PDB code: 1k8r; yellow), RAP1A-CRAF (PDB code: 1gua; lime), KRAS-ARAF (PDB code: 2mse; magenta), HRAS-RALGDS (PDB code: 1lfd; cyan), HRAS-PI3K (PDB code: 1he8; green), HRAS-PLCε (PDB code: 2c5l; orange), HRAS-RASSF (PDB code: 3ddc; blue), HRAS-GRAB14 (PDB code: 4k81; brown), were overlaid in ribbon presentation. Additional properties outside the interaction interface (box) are indicated.
Fig 4.
RAS-effector interaction hotspots.
(A) Interaction matrix of RAS isoforms and effector proteins. Interaction matrix is launched to demonstrate interaction residues in all available structures (see Fig 3 and S4 Fig). Left and upper parts comprise the amino acid sequence alignments of the RAS proteins and the effector domains, respectively. Each element corresponds to a possible interaction of RAS (row; HRAS numbering) and effector (column; CRAF numbering) residues. As indicated, interaction matrix represents five main regions, which cover the main interacting interfaces. (B) The five main regions, comprising the main hotspot for the RAS-effector interaction, are highlighted as ribbon and surface representations in the corresponding colors for the structures of HRAS-PLCε (PDB code: 2C5L) and HRAS-CRAF (PDB code: 4G0N). Key amino acids which are highlighted by colored background in A are indicated on the structures as well.