# Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers
## 2 Results
The underlying mechanism of the SOScat-assay is that mant-GDP is exchanged by non-fluorescent GDP in the presence of Ras-GEF SOScat. By varying the BPNH2 concentration, a K\({}_{\rm D}\) of 0.20 \(\pm\) 0.10 mM could be extracted from this SOScat-assay (Figure 4). This K\({}_{\rm D}\) value for BPNH2 is slightly lower than for BPAF, which can be attributed to the amino groups as hydrogen bond donors [21]. Figure 3: Overall view of the NMR chemical shift perturbation observed for K-Ras4B bound to GDP upon titration with Bisphenol AF (**upper** panel) and BPNH2 (**lower** panel), ranging from the black reference to a ratio of 1:25, shown in magenta. The enlarged panels illustrate residues G60, T74, and G75. The titrations were performed as previously published [9]. In order to decrease solubility artefacts, the different ligands were titrated in the same % \(v/v\) steps, using different stock concentrations. Additionally, we performed a GDI-assay that observes inhibition of nucleotide dissociation [18; 20] (Figure S7). Hitherto, this method was used to measure complex formation between Ras and its effectors. Surprisingly, while BPAF showed no or only a very small effect, BPNH\({}_{2}\) exhibited a significant GDI-effect with a K\({}_{\rm D}\) value of 0.34 \(\pm\) 0.02 mM, which is in good agreement with the value derived from the SOS\({}^{\rm cat}\)-assay and the NMR experiments (Figure 3, Figure 4 and Figures S1 and S7; Table 1 and Table S1). Taken together, these three independent biophysical and biochemical assays show that both BPNH\({}_{2}\) and BPAF interfere with the SOS\({}^{\rm cat}\) mediated nucleotide exchange, but only BPNH\({}_{2}\) exhibits a functional GDI effect on K-Ras4B (Figure 4 and Figure S7). Consequently, the characteristic line broadening of the NBD cluster resonances and the inhibition of intrinsic nucleotide release are caused by the introduction of amino groups at both phenolic moieties. It is interesting to note that the characteristic line broadening for switch I and II of GppNHp-loaded GTPase caused by micro- to millisecond dynamics does not occur for GDP-bound K-Ras4B when complexed with a bisphenolic compound. Apparently, the amide resonances of the switch regions in the GDP/K-Ras4B complex experience fast rather than intermediate exchange upon binding of bisphenols (Figure 5). The amide resonance intensity of Y157 from Ras-GTP is known to gradually decline while the corresponding resonance from Ras-GDP increases accordingly as GTP hydrolysis proceeds [22]. The proton-amide chemical shift of Y157 clearly shows that the compounds gradually shift the conformational equilibrium via intermediate positions from the resonance of the inactive to the active form (Figure 5). The amide resonances of residues I24, Q25, D57, G75, A83, I84, N86-S89, H94, G115, C118, Q129-Q131, I139, S145, T148, and F156-T158 in K-Ras4B bound to GDP experience chemical shift perturbations towards resonance frequencies of K-Ras4B bound to GppNHp upon binding of BPAF (Figure 5 and Figure S5). These residues are not located close to the BPAF binding pocket of K-Ras4B but cluster around the switch I and II regions, the region next to the bound GDP as well as neighbouring helices (Figure 5). This suggest that binding of BPAF to GDP-loaded K-Ras4B allosterically induces to a certain extent a conformation that resembles the active, GppHp-bound form of K-Ras4B. Quantitatively, BPNH\({}_{2}\) binding shifts the amide NMR resonance of Y157 to approx. 64% of the Y157 resonance frequency in the active, GppNHp-bound state (Figure S6). For the saturated BPAF-, BPA-, and BPS-protein complex this effect is less pronounced at 58%, 49%, and 20%, respectively. We note that these different levels of allosterically activated GDP-bound K-Ras4B induced by bisphenols correlate reasonably well with K\({}_{\rm D}\) values determined by NMR and IC\({}_{50}\) values extracted from a MTT assay (Figure 5 and Figures S6 and S7).