## 3 Results
### Allosteric Communication across the Ras-Raf-RBD_CRD Dimer Complex
The remaining two means for information transfer directly involve the Raf-CRD, with one utilizing the Raf-CRD's close association with loop 8, creating paths that wrap around the Ras active site (Figure 3c); and the other alternatively using the interconnectivity between the Raf-CRD and interswitch region to link the two Ras protomers again through loop 3 and \(\lx@sectionsign\)6 (Figure 3d). The allosteric connections linking residues D113 at the Galectin-binding pocket of each Raf-RBD molecule in the dimer, which also includes residue D117 [13] (Tables S5-S7), suggest a model where Ras-Raf-RBD_CRD dimers couple with Galectin dimers to form a higher-order macromolecular platform involved in signal amplification and kinetic proofreading [8] (Figure 3e), similar to that described for the LAT/Grb2/SOS system [58]. This model is consistent with the functional importance of Galectins 1 and 3 for signaling through the MAPK pathway [15; 16; 59; 60] and with results from single molecule tracking experiments correlating immobile species of Ras observed in live cells to active signaling through the Ras-Raf-MEK-ERK pathway [61; 62; 63]. Our molecular dynamics simulation data suggest that the interconnectivity between the Raf-CRD and the interswitch region of one Ras molecule and loop 8 of the other, in the context of its dynamic nature, optimally positions the Raf-CRD to diversify the paths of allosteric communication across the Ras-Raf complex linking the Galectin-binding sites at the two ends of the dimer, not only by further bridging the two Ras molecules in the Ras dimer, but also serving as a link between the Raf-RBD and second Ras molecule in the complex, with a direct connection to the Ras active site. Simulations of the KRas-CRaf-RBD dimer complex modelled on the membrane demonstrate that this complex is flexible at the interface [8]. The Raf-CRD may accommodate the complex's dynamic nature by providing alternative routes of communication between the two Galectin-binding regions at opposite ends of the dimer. The prominence of each of the alternate allosteric paths facilitated by the CRD may be modulated by isoform-specific residues and sensitive to oncogenic mutations in the active site. Figure 3: Dynamical network analysis performed for HRas–CRaf-RBD_CRD dimer simulations. (**a**) Allosteric paths calculated between D113 and D113’ of the Raf-RBD molecules. (**b**) Close-up view showing path for intermolecular information involving Ras loop 3/86 (orange) and Ras \(\alpha\)5/\(\beta\)6 (red) across the Ras dimerization interface. (**c**) Close-up view showing path for intermolecular information involving Raf-CRD/Ras loop 8 (purple). (**d**) Close-up view showing path for intermolecular information involving Raf-CRD/Ras interswitch region (blue). (**e**) Proposed Ras–Raf–Galectin assemblies adapted from [PERSON] et al. [8] with inclusion of the Raf-CRD. Galectin (grey) (PDB ID 3W58) and Raf-kinase (purple) (PDB ID 6Q0J) were downloaded from the Protein Data Bank. The scaffold protein 14-3-3 stabilizes the Raf kinase dimer (not shown). Given that the structure of the CR2 is unknown, it is also possible that kinase domains from distinct Ras–Raf-RBD_CRD dimers bridged by Galetin come together to form the activated Raf dimer.