# Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers
## 3 Discussion  
Taken together, we observe for all bisphenols tested in this study, ranging from BPF to BPNH\({}_{2}\), direct binding to a well-characterized binding pocket between Switch I and Switch II and an allosteric effect on the NBD of K-Ras4B. However, only for BPNH\({}_{2}\) do we observe line broadening for the NBD residues G10, G13, and G60 (Figure 3). Also, T35 exhibits similar chemical shift perturbation changes upon titrating BPNH\({}_{2}\) to K-Ras4B. This line broadening was not observed for the switch regions, what would be a direct indicator for active K-Ras4B. In addition, BPNH\({}_{2}\) executes a GDI effect on K-Ras4B and binding of SOS to the BPNH\({}_{2}\)-Ras(GDP)-complex is impaired (Figure 4 and Figure S7). Ras adopts at least two main conformational states in accordance to the binary switch on/off-model. In the triphosphate-bound conformation of Ras, at least two states, termed T1/T2, exist. Remarkably, only T2 is capable of binding to effectors [(23)]. Furthermore, the GDP-bound form of Ras is characterised by heterogeneous states, denoted as polysterism [(24)]. We hypothesise that binding of bisphenolic ligands to K-Ras4B alter its conformational ensemble while still bound to GDP, favouring a state in-between the active and inactive one. Our NMR data clearly show that BPAF can antagonise the GDP-bound K-Ras4B/SOSO\({}^{\text{cat}}\) interaction and that bisphenols act as non-covalent allosteric agonists on GDP-bound Ras (Figures 1, 2 and 5). This agonistic effect of bisphenols on Ras in vivo under physiological conditions is corroborated by G-LISA Ras activation assay and western blot analysis, respectively (Figure 6). Taken together, BPAF can activate the c-Raf kinase pathway and partly PI3K signalling whereas BPNH\({}_{2}\) only acts on the latter under physiological conditions in HEK293T cells. This is presumably achieved by utilising the different affinities over a broad range of Ras-family members towards their effector molecules [(18; 25)]. It is interesting to note that a similar yet not identical mechanism of activation has also been observed for other small GTPases, e.g., Rab1b, a main regulator of membrane trafficking [(26)]. Unlike the non-covalent allosteric agonistic mode  
Figure 6: Level of BPAF-induced activation reported in %. Serum-starved HEK293T cells were treated with DMSO (Ctrl), BPAF [20 \(\upmu\)M] and BPNH\({}_{2}\) [33 \(\upmu\)M] in a time-dependent manner (1, 2 and 4 h). EGF-stimulation [10 ng/mL] was carried out for 10 min. Thereafter, cells were lysed and subjected for G-LISA Ras Activation Assay Biochem Kit shown in the upper left corner and to immunoblot analysis (for pERK/pAKT in relation to total ERK/AKT) shown on the right. The graph is presented as mean \(\pm\) SEM (\(n\) = 2, each experiment was carried out in triplicate; * \(p\) < 0.05; *** \(p\) < 0.001). The quantification of the pERK versus total ERK ratio is shown in the lower left corner. To this end, serum-starved HEK293T cells were treated with DMSO (Ctrl) or BPAF [20 \(\upmu\)M] for 4 h. Then, cells were lysed and subjected to immunoblot analysis (for pERK in relation to total ERK). Quantification is based on three independent experiments and is presented as mean \(\pm\) SEM (* \(p\) < 0.05). of action of bisphenols on Ras, Rab1b is covalently AMPylated by the Legionella effector protein DrrA at tyrosine 77 (Y71 in Ras proteins) and this modification stabilises the active state, as shown by molecular dynamics (MD) simulation [27]. As suggested by [PERSON] et al., this tyrosine is also very important for Ras to interact with ligands, as the phenolic side chain has to change its position in order to create a cavity suitable for ligand binding [6]. It is believed that the nucleotide binding state of the Ras protein controls the positioning of the switches. Further, MD simulations suggested, that the 3D orientation of the Switch II is directly affected by the interplay between G60 and \(\gamma\)-phosphate [28].