The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

<div><p>RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our <i>in vitro</i> data with <i>in silico</i> data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS <i>versus</i> RRAS isoforms with effectors.</p></div

Equilibrium dissociation constants for RAS-effector interaction determined Fluorescence polarization.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167145#pone.0167145.s002" target="_blank">S1 Fig</a> (B) Evaluated equilibrium dissociation constants (K_d) 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.</p

Domain organization of RAS effectors and different proteins used in this study.

<p>(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 <i>domain;</i> 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.</p

Register of dissociation constants (K_d) determined for the RAS-effector interactions.

<p>Register of dissociation constants (K_d) determined for the RAS-effector interactions.</p

RAS-effector interaction hotspots.

<p>(A) Interaction matrix of RAS isoforms and effector proteins. Interaction matrix is launched to demonstrate interaction residues in all available structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167145#pone.0167145.g003" target="_blank">Fig 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167145#pone.0167145.s005" target="_blank">S4 Fig</a>). 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.</p

AUC-SV data for NPM1^FL, NPM1^OD, and US11^FL, respectively.

<p>MW, molecular weight; S20,w (S), sedimentation rate at 20°C; f/f0, frictional coefficient. In all three cases the values refer to a single, dominant species, which represented more than 90% of the sample.</p><p>AUC-SV data for NPM1^FL, NPM1^OD, and US11^FL, respectively.</p

NPM1 association with HSV-1 US11 in the cell.

<p>(A) Nucleolar colocalization of endogenous NPM1 with myc-US11. Confocal images of HeLa cells transfected with myc-US11 were obtained by staining endogenous NPM1 (Mouse anti-NPM1 (ab10530)), myc-US11 (anti-myc antibody), and filamentous actin (rhodamine-phalloidin). For clarity, a boxed area in the merged panel shows colocalization of NPM1 and US11 in the nucleolus as pointed by arrows. Scale bar: 20 μm. (B) Myc-US11 associates with endogenous NPM1 in COS-7 cells. NPM1 was co-immunoprecipitated with myc-US11 overexpressed in COS-7 cells using anti-myc antibody. A normal Rabbit IgG and sample without antibody were used as IP controls. Input, 5% of total cell lysate; IP, immunoprecipitation; IB, immunoblotting. (C) Myc-US11^FL displayed an interaction with NPM1^FL. Myc-US11^FL was pulled down with the GST-fusion NPM1^FL, but not with GST, which was used as a negative control. Samples prior pull-down (PD) analysis were used as input control.</p

Direct NPM1 interaction with HIV-1 Rev.

<p>(A) Qualitative interaction analysis by GST pull-down assay and subsequent CBB staining. NPM1 FL, OD and HRBD, but not RBD, displayed a selective interaction with HIV-1 Rev (upper panel), which was also observed after an RNase A treatment (lower panel). (B) Quantitative interaction analysis by ITC. The binding parameters for the interaction between NPM1^FL and Rev were obtained using ITC. Titration of NPM1^FL (750 μM) to Rev^FL (35 μM) showed an exothermic response (negative peaks) indicating that Rev selectively interacts with NPM1^FL. The upper graph shows calorimetric changes plotted versus the time and the lower graph represents the changes in temperature according to the molar ratio of the interacting proteins. (C) No interaction was observed in a control experiments by titrating NPM1^RBD (300 μM) to Rev^FL (30 μM).</p

CIGB300 treatment interferes with HIV-1 production.

<p>CIGB-300 treated or untreated HOS.CD4.CXCR4 cells were infected with NL4.3 virus at an MOI of 1. Culture supernatant was collected 48 and 72 h post infection and virus titer was determined. The figure shows one representative experiment out of four, in which virus quantification was performed by TZM-bl cell titration. Values are the means ± S.D. of three measurements. Statistical significance (P) was calculated by the Student`s t-test: ***P<0.002; **P<0.02.</p

The synthetic peptide CIGB-300 competes with Rev and US11 by binding NPM1^OD with high-affinity.

<p>(A) CIGB-300 consists of the cyclic P15 (blue) and the Tat (purple) peptides, and labeled with fluorescein (green; FITC). (B) Fluorescence polarization experiments conducted by titrating increasing amounts of NMP1 variants, Rev, US11, and GST to 0.1 μM FITC-labelled CIGB-300 (f CIGB-300). A high affinity interaction with the peptide was only observed for NPM1^FL and NPM1^OD, resulting from an increase of polarization, but not for Rev, US11, GST, and the other NPM1 variants. (C-D) Contrary to US11, Rev only displaced NPM1^OD from its fCIGB-300 complex. Displacement experiments were performed by adding increasing amounts of Rev or US11 to the NPM1^FL-fCIGB-300 complex (C) or to the NPM1^OD-fCIGB-300 complex (D). (E) A proposed NPM1^OD-CIGB-300 docking model of pentameric NPM1^OD structure in the complex with CIGB-300. Cyclic part (blue) and basic part (purple) of the peptide shown as sticks and ribbons wraps around several monomeric units of NPM1 represented by surfaces in different colors shown in top view (left), rotated orientation (middle), and the bottom view (right).</p