# Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers
## 2 Results
Molecular dockings using the HADDOCK software suite [15] suggest that BPAF binds at the interface of SOS\({}^{\rm cat}\) and K-Ras4B and sterically antagonists Ras/SOS\({}^{\rm cat}\) protein complex formation, neighbouring amino acids Y64, M67, and Y71 that are known as the hydrophobic anchor of the Ras-SOS\({}^{\rm cat}\)-interaction [16]. Additionally, reorganisation of switch II is fundamental to the interaction between K-Ras4B and SOS\({}^{\rm cat}\)[16], and both mechanisms are apparently affected by BPAF binding. Since Ras isoforms share a high sequence similarity, we also tested H-Ras and N-Ras for their potential to interact with BPAF. For H-Ras and BPAF, a similar pattern of CSPs was observed and the NMR-based titration experiment yielded a K\({}_{\rm D}\) of \(0.40\pm 0.02\) mM. For N-Ras (1-170), we also observed a similar pattern of CSPs. In addition, the K\({}_{\rm D}\) was in the same range (0.77 \(\pm\) 0.02 mM). These results show that all three Ras-isoforms interact equally with BPAF and that, at least for H-Ras and K-Ras4B, the HVR-region does not affect binding of BPAF. Rap-1A, which shares a sequence similarity of 50% with Ras, was also tested. It is known, that the Ras/Rap effector specificity is determined by charge reversal [17]. When Rap-1A was titrated with BPAF, CSPs up to a BPAF concentration of 5 mM could not be detected (Figure S2). Obviously, numerous changes of binding pocket residues (for example T74 to N74) prevent Rap-1A from binding to BPAF, in accordance with GTPase-effector specificity. Apparently, BPAF is able to select K-Ras4B, H-Ras, and--to a slightly lesser extent--N-Ras over Rap-1A. Noteworthy, Ras-family members, such as H-Ras, Rap-1A, and R-Ras, exhibit different affinities over a broad range towards the effector molecules c-Raf kinase and Ral guanine nucleotide exchange factor (RGF), for instance [17; 18]. The aromatic phenyl rings of the BPAF analogue BPNH\({}_{2}\) are amino-substituted at the _meta_-position and when titrated to  
Figure 1: Definition of the different binding clusters: the 1 st (ligand) ring (L6, I55, L56, T74, G75), the buried hydrophobic cluster (L79, C80, I93), and the NBD (nucleotide binding cluster, D57 and G60), and their location within the GTPase fold. K-Ras4B, an intermediate-exchange of resonances for the binding pocket residues (L56, D57, T74, and G75) was observed at 600 MHz \({}^{1}\)H Larmor frequency, contrary to a fast exchange detected for BPAF. This indicates a change in binding kinetics (Figure 3). In addition, the NBD cluster (G10, G13, D57, and G60) exhibits line broadening of NMR resonances. Threonine 35, an amino acid which is next to G60 and fundamental to the loaded spring model [19], exhibits a change in chemical environment that corresponds to fast/intermediate exchange upon ligand binding. Figure 2: Competitive titration of \({}^{15}\)N-enriched GDP-bound K-Ras4B with \({}^{14}\)N SOS\({}^{\text{cat}}\) and BPAF at 600 MHz and 298 K. Different 2D \({}^{1}\)H-\({}^{15}\)N HSQC NMR spectra of this titration are shown, starting with the K-Ras4B GDP protein only (reference in black). In dark and lighter blue spectra with the GEF-protein SOS\({}^{\text{cat}}\) added (molar ratios of 1:1 and 1:2) are depicted. The resulting line broadening of resonances is clearly visible. In green, the ligand BPAF is added in a molar ratio of 1:2:0.5. In orange and pink, the 1:2:2 and 1:2:4 molar ratio titration steps are shown. The recovery of backbone amide proton NMR resonances is indicated by dashed yellow circles. It is important to note that the recovered resonances of residues from the binding pocket exhibit chemical shift perturbations compared to ligand-free 2D \({}^{1}\)H-\({}^{15}\)N HSQC spectra of \({}^{15}\)N-enriched GDP-bound K-Ras4B that match those observed during the titration of GDP-bound K-Ras4B with BPAF alone. Furthermore, we performed SOScat-catalysed guanine nucleotide exchange (SOScat-assay) and guanine nucleotide dissociation (GDI-) assays, that both exploit the protein-bound fluorescent mant-nucleotide [20].