Mutation of basic residues in helix α4 or the HVR has differential effects on the FRET vectors of GTP- and GDP-H-ras. MD simulations (Gorfe et al, 2007b) suggest GTP- and GDP-H-ras have different membrane interactions and orientations of the G-domain with respect to the plane of the membrane. (A) The GTP conformation is stabilized by membrane contacts of R128 and R135 on helix α4. (B) These contacts are lost in GDP-H-ras, which is stabilized by contacts of residues R169 and K170 in the HVR. Phosphorous atoms of lipid head groups of the inner membrane leaflet are shown in grey, the outer leaflet is shown as a grey line (not to scale) and H-ras lipid anchors are in light blue. Important basic residues in H-ras are shown in dark blue and acidic residues in red. (C, D) The matrices show eight-tuple FRET vectors for GTP- and GDP-H-ras with mutations at the indicated residues. Each FRET vector is the set of E_max values of a mCFP-tagged H-ras mutant (blue) and eight mCit-tagged nanodomain markers (yellow). The matrix shows E_max values±s.e.m. and number of independent experiments in brackets. E_max values of each mutant–marker pair were compared with the corresponding value in the cognate wild-type background (grey FRET vectors) using t-tests; significant differences are shown by green shading. The matrices show different patterns of altered E_max values in the GTP and GDP background.

The novel membrane-orientation switch regulates H-ras interaction with C-Raf and MAPK signaling. (A) HEK293 cells transiently expressing mGFP-tagged H-rasG12V mutants with or without an excess of mRFP-RBD were imaged using FLIM. The mean mGFP donor fluorescence lifetime (±s.e.m.) was determined for multiple ROIs and data pooled from three independent experiments. The number of ROIs analysed is given in brackets. Statistically significant differences from the lifetime of mGFP-H-rasG12V coexpressed with mRFP-RBD were assessed using t-tests (^*P<0.05, ^**P<0.01, ^***P<0.001). Whereas only the lifetime of mGFP-H-rasG12V in the absence of mRFP-RBD is shown, the lifetimes of all other mGFP-H-rasG12V mutants expressed alone were the same (Supplementary Figure S2C). (B) BHK cells transiently expressing fluorescently tagged H-rasG12V (GTP-H-ras) with the specified mutations were assayed for MAPK activation by quantitative immunoblotting for ppERK. The figure shows mean ppERK levels (±s.e.m.; n=3). ^*Significant (P<0.05) increase in MAPK activity compared with non-mutated H-rasG12V. (C) Intact 2D plasma membrane sheets prepared from BHK cells expressing mGFP-H-ras (GDP-H-ras) or mGFP-H-rasG12V proteins with the specified mutations were immunogold labelled and imaged by EM. The graphs show a statistical analysis of the resulting immunogold point patterns as weighted mean K-functions calculated from n=16–27 membrane sheets. The gold labelling density for these experiments was 281–478/μm². Nanoclustering is quantified by the extent of the positive deflection of the L(r)–r curve out of the confidence interval for a random pattern (±1). The radius of the nanoclusters is correlated with the radius at which the L(r)–r deflection is maximum. Using parametric bootstrap tests, we detected no significant differences in either of these nanoclustering parameters from control GTP- or GDP-H-ras point patterns for any of the mutants that were evaluated.

FRET vectors describe the lateral segregation of Ras proteins. (A) The extent of FRET between pairs of mCFP (blue squares) and mCit (yellow squares)-labelled Ras probes and complementary labelled nanodomain markers was measured. The nanodomain probing constructs were either Ras-derived membrane anchors or full-length H-Ras mutants (Supplementary Figures S1C and S4). High FRET values are expected for proteins that co-cluster, whereas a random distribution will result in no or very little FRET. In addition, markers of different lengths enable detection of conformational changes (Supplementary Figure S1B). The nanodomain markers C and D indicate that a set of nanodomain markers is used sequentially to characterize the lateral segregation of the Ras-derived probes by a set of FRET values. (B) Amino-acid sequences of Ras membrane anchors used for FRET experiments. (C) All mCFP/mCit-tagged proteins showed predominant localization to the plasma membrane and were homogeneously distributed when imaged by confocal microscopy (Supplementary Figure S4). We, therefore, increased the throughput of the analysis of >140 FRET pairs, by measuring fluorescence signals in a cytometer. The dependence of the FRET efficiency, E, on the normalized acceptor surface concentration, cA, at ∼1:1 donor–acceptor ratio was analysed using equation (1) (red curves), which yielded the characteristic FRET value, E_max. The E_max for most FRET pairs was higher than expected for randomly distributed donor and acceptor species (lower panel, calculated curve in blue), indicative of nanoclustering (Abankwa and Vogel, 2007). Data points are calculated from single cells, the example plots shown are mCFP-H-rasG12V/mCit-tH (upper panel) and mCFP-K-rasG12V/mCit-tH (lower panel). (D) The sample matrix shows E_max values (±s.e.m. and number of independent experiments n) for Ras membrane anchor probe and marker FRET pairs. The lateral segregation of each Ras membrane anchor probe is described by the FRET vector, given in this example by the set of three E_max values in each row. (E) FRET vectors can be plotted in a nanodomain marker 'space'. The direction of a vector describes the lateral segregation of a probe. Thus, the more similar the direction of FRET vectors, the more similar is the lateral segregation of the marker probes. The colouring matches that in (D).

A membrane-orientation switch region in H-ras. A combination of MD simulation and structural analysis (Supplementary Figure S2A) suggests operation of a membrane-orientation switch region in H-ras that comprises the β2–β3 loop, helix α5 and the HVR. (A, B) The models for GTP- and GDP-H-ras show networks of salt bridges between basic and acidic residues, which we propose are involved in the structural changes that reorient H-ras after GTP loading. Colouring is as in Figure 2A. Lipid molecules are represented as thin sticks. (C, D) The two matrices show FRET vectors of GTP- and GDP-H-ras with alanine substitutions at residues involved in the membrane-orientation switch. E_max values are given±s.e.m., and number of independent experiments in brackets. E_max values of each mutant–marker pair were compared with the corresponding value in the cognate wild-type background (grey FRET vectors in Figure 2C and D) using t-tests, significant differences are shown as green shading. (E, F) Correlation maps of all GTP- and GDP FRET vectors in C, D and Figure 2C and D. In these maps, mutated residues are arranged in a similar spatial layout as they appear in the H-ras structure, mutated residues are connected by lines if the correlation coefficient is >0.6, the thickness of the line is proportional to the correlation coefficient (given in Supplementary Figure S2B). Hence, mutants linked by thicker lines cause similar perturbations in the parent FRET vector. Changes in the correlation maps are suggestive of actual structural rearrangements, possibly linking regions of correlated motion. The network in E clearly shows that residues in the switch region stabilize a conformation that realizes the membrane contact of helix α4. The network in F reveals complex rearrangements of linkages in the switch region that correspond to a different stabilization of the GDP-H-ras conformation; the strongest participation is that between D47 and R169/K170.

A balance model identifies another level of regulating Ras signal output. The balance model introduces the orientation of the G-domain with respect to the plasma membrane as an additional determinant of signalling specificity among Ras isoforms. The balance is regulated by the membrane-orientation switch region, which comprises residues in the β2–β3 loop and helix α5, and is indicated by the black fulcrum. MD and FRET analysis of H-ras show that for inactive, GDP-bound Ras (red) contacts of residues in the hypervariable region (HVR) prevail (i) so the balance is shifted to the HVR. Upon GTP loading, Ras is reoriented by interactions of helix α4 with the membrane (ii) so the balance is shifted to helix α4. In the context of this balance model, different Ras isoforms of the Ras subfamily may adopt different preferred orientations with respect to the membrane depending on the precise combination of residues present in the HVR and helix α4 (Supplementary Figure S3). Schemes (iii) and (iv) show examples of the membrane-interaction balance being shifted more and less towards helix α4, respectively. We speculate that such submembrane orientations are an important determinant for the propensity of GTP-loaded Ras proteins to interact with other membrane-associated proteins, such as its activity modulators (e.g. galectins) and other effectors (e.g. PI3 kinase).