3.1 Phosphorylation in the accessory domain exposes the nuclear export signal sequence
Five trials of 100-ns MD simulations for each system (Nap1-nonP and Nap1-P) were performed to evaluate the phosphorylation effect in the accessory domain. Through these MD simulations, we observed that the accessory domain fluctuated more heavily than the other regions on yNap1. In terms of their root-mean-square fluctuation (RMSF) profiles (Figure 2), the two systems had no obvious differences between them, indicating that phosphorylation in the accessory domain did not change the overall fluctuation of yNap1.
Next, we focused on the location of the accessory domain in relation to the NES before and after phosphorylation. Figure 3 shows the representative structures of Nap1-nonP and Nap1-P. In Nap1-nonP, the accessory domain clearly masked the NES (Figure 3A), whereas in Nap1-P, it was dislocated from the sequence (Figure 3B). To evaluate this dislocation quantitatively, we determined the exposure of the NES as a measure of the SASA (Figure 4). The median of the SASA distribution of Nap1-P was greater than that of Nap1-nonP, indicating that phosphorylation in the accessory domain increases the exposure of the NES and its accessibility to exportin, thereby promoting the export of yNap1 from the nucleus.