K-Ras4B is a protein involved in cell signaling. By switching between active GTP-bound and inactive GDP-bound conformation, it functions as a molecular switch, regulating the intensity and the timescale of cellular signals leading to various cellular responses such as cell growth, differentiation and inhibition of apoptosis. K-Ras4B is one of the most frequently mutated proteins in human cancers. Despite decades of efforts, the success in treating cancer by inhibiting K-Ras4B has been limited. For further development of K-Ras4B inhibitors a deep understanding of the biological and chemical mechanisms of its actions is required.
In its active form, K-Ras4B is anchored to the plasma membrane through a disordered C-terminal region. The catalytic domain interacts with the membrane transiently however, it has been shown, that the catalytic domain interacts with the membrane preferentially in a limited number of distinct orientations relative to the membrane. In some of the orientations, the interaction with proteins, important for signalling function of K-Ras4B, may be hindered by the membrane, which suggests, that the orientation could influence the activity of the protein. There has been a significant effort in characterizing the most populated orientations and their transitions, however, the effects of different orientations on the internal dynamics of the catalytic domain have not yet been investigated.
In this study we performed molecular dynamics simulations with the corresponding analysis of structure and dynamics of K-Ras4B in wild type form and with an oncogenic mutation G12D in GTP- and GDP-bound states in water and in two orientations, which have been experimentally shown as the two dominant orientations. We have corroborated the experimentaly observed fact that in G12D-K-Ras4B the stability of the orientation with parallel helices (H2) relative to the membrane compared to the orientation with perpendicular helices (P1) is increased in G12D-K-Ras4B relative to the wild type. We observed conformational transitions that could be favorable GDP-GTP exchange in K-Ras4B-GDP in H1. Using quasiharmonic analysis we have shown, that the vibrational entropy of membrane-bound protein is increased, which could facilitate the conformational changes, important for protein function. We have observed low-frequency normal modes, which are specific for membrane-bound protein and differ between different orientations. These modes feature displacements of atoms in regions, important for interaction with K-Ras4B binding proteins and might indicate preferences for specific functional conformational changes on the membrane.
Our results give insight into the effect of the membrane orientation on dynamics of K-Ras4B, which could contribute to the development of K-Ras4B inhibitors that would act by modulating the orientational preferences towards inactive orientations.
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