# Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers
## 2 Results  
AFX [4-(Trifluoromethyl)phenol] was tested in order to evaluate the effect of a single ring ligand on K-Ras4B (Table 1). Using multi-dimensional NMR spectroscopy, we detected significant chemical shift perturbations (CSPs) induced by AFX for amino acids L6, I55, L56, D57, T74, and G75 of \({}^{15}\)N isotopically-enriched K-Ras4B. However, a K\({}_{\rm D}\) value could not be determined precisely (K\({}_{\rm D}\) > 50 mM). Next, we tested the simplest structural bisphenolic ligand, which is BPF carrying two hydrogens instead of two methyl groups in BPA located outside the AFX binding pocket of K-Ras4B, followed by other bisphenol derivatives (Table 1 and Table S1). Interestingly, amino acids that experienced CSPs were, in part, neither surface-exposed, nor close to the AFX binding pocket. Based on their location in the canonical GTPase fold, the affected residues can be grouped into three clusters (Figure 1). The first cluster comprises amino acids that contact the first phenyl ring of BPA (L6, I55, L56, T74, G75). The second cluster mainly consists of hydrophobic residues (V7, V8, V9, L79/C80, I93), which sense the ligand binding information indirectly through L6. Thirdly, the nucleotide binding domain (NBD)-cluster is made of the amino acids G10, G13, D57, and G60 that are triggered by the interaction between the amide group of L56 and the ligand. The same binding pattern for these three clusters is also observed for BPE, which contains one hydrogen atom and one methyl group at the central carbon atom and exhibits a K\({}_{\rm D}\) of 6.5 \(\pm\) 0.7 mM. Bearing two CH3 groups, BPA yields a K\({}_{\rm D}\) of 0.6 \(\pm\) 0.2 mM [9] and mainly exhibits the same pattern as observed for BPF and BPE. In the case of BPB, which contains one methyl and one ethyl substitution, we observed a deterioration of the K\({}_{\rm D}\) to 3.6 \(\pm\) 0.7 mM. Apparently, the ethyl group is sterically too demanding and prevents the exact ring phenyl orientation required for tighter binding. This observation is in agreement with previously published results for BPS [10]. Substituting protons with fluorine has been shown to potentially \begin{table}
\begin{tabular}{|p{113.8 pt}|p{113.8 pt}|p{113.8 pt}|p{113.8 pt}|} \hline AFX & BPF & BPE \\ HO & HO & HO \\ \hline \(\text{K}_{\rm D}\) = ND & \(\text{K}_{\rm D}\) = 14 \(\pm\) 2 mM & \(\text{K}_{\rm D}\) = 6.5 \(\pm\) 0.7 mM \\ \hline BPA & \(\text{BPP}\) & \(\text{H}_{\rm 3}\text{C}\) & \(\text{HP}\) \\ HO & HO & HO & HO \\ \hline \(\text{K}_{\rm D}\) = 0.6 \(\pm\) 0.2 mM & \(\text{K}_{\rm D}\) = 3.6 \(\pm\) 0.7 mM & \(\text{K}_{\rm D}\) = 0.4 \(\pm\) 0.1 mM \\ \hline BPS & \(\text{BPNH2}\) & \(\text{K}_{\rm D}\) = 0.4 \(\pm\) 0.1 mM & Low solubility \\ \hline \end{tabular}
\end{table} 
Table 1: Bisphenols tested in this NMR study, with varying bridging moieties at the central sp3-hybridised carbon atom. AFX was used to characterize the binding of one phenolic ring to K-Ras4B. ND stands for not determined. alter molecular conformation and to specifically enhance affinity for proteins [14]. In line with these observations, the presence of two fluoromethyl groups in BPAF yields a slightly lower K\({}_{\rm D}\) value of 0.35 \(\pm\) 0.02 mM (Table 1 and Table S1; Figures S1-S4). Since BPAF exhibits the largest affinity for K-Ras4B, we tested as to whether BPAF could antagonise the interaction between K-Ras4B and SOS\({}^{\rm cat}\). Thus, we carried out a titration of \({}^{14}\)N SOS\({}^{\rm cat}\) to \({}^{15}\)N-K-Ras4B to create a complex of around 80 kDa, which leads to a broadening of NMR resonances presumably due to rotational correlation effects. This can be observed at a 1:1 ratio and is further pronounced when more SOS\({}^{\rm cat}\) is added (Figure 2 and Figure S3). When BPAF was titrated to this complex, previously vanished resonances could be recovered at the frequencies specific for the bisphenol-bound K-Ras4B protein. Thus, BPAF can antagonise the interactions between GDP-bound K-Ras4B and SOS\({}^{\rm cat}\).