Garcia, A. E. | bioRxiv Science

bioRxiv2019

Presence or absence of Ras-dimerization shows distinct kinetic signature in Ras-Raf interaction

In eukaryotes, MAPK pathways play a central role in making several important cellular decisions, including cell proliferation and development of an organism. Ras, a small GTPase, interacts with the protein Raf to create activated Ras-Raf complex (Raf dimer) that activates the downstream effectors in the ERK pathway, one of the many MAPK pathways. Malfunctioning Ras-Raf \"switches\" cause almost 30% of all known cancer. Hence, understanding Ras-Raf interaction is of paramount importance. Despite decades of research, the detailed mechanism of Ras-Raf interaction is still unclear. It has been hypothesized that Ras dimerization is necessary to create the activated Raf dimer. Although there are circumstantial evidences supporting the Ras dimerization hypothesis, direct proof of Ras dimerization is still inconclusive. In the absence of conclusive direct experimental proof, this hypothesis can only be examined through indirect evidences of Ras dimerization. In this paper, using a multi-scale simulation technique, we provide multiple criteria that distinguishes an activation mechanism involving Ras dimerization from another mechanism that does not involve Ras dimerization. The provided criteria will be useful in the investigation of not only Ras-Raf interaction but also other two-protein interactions.

biophysics↗

bioRxiv2019

The plasma membrane as a competitive inhibitor and positive allosteric modulator of KRas4B signaling

Mutant Ras proteins are important drivers of human cancers, yet no approved drugs act directly on this difficult target. Over the last decade, the idea has emerged that oncogenic signaling can be diminished by molecules that drive Ras into orientations in which effector binding interfaces are occluded by the cell membrane. To support this approach to drug discovery, we characterize the orientational preferences of membrane-bound K-Ras4B in 1.45 milliseconds aggregate time of atomistic molecular dynamics simulations. Individual simulations probe active or inactive states of Ras on membranes with or without anionic lipids. We find that the membrane orientation of Ras is relatively insensitive to its bound guanine nucleotide and activation state but depends strongly on interactions with anionic phosphatidylserine lipids. These lipids slow Ras translational and orientational diffusion and promote a discrete population in which small changes in orientation control Ras competence to bind multiple regulator and effector proteins. Our results suggest that compound-directed conversion of constitutively active mutant Ras into functionally inactive forms may be accessible via subtle perturbations of Ras orientational preferences at the membrane surface.\n\nStatement of SignificanceMutations that lock Ras proteins in active states can undermine cellular decision making and drive cancer. Because there are no drugs to deactivate Ras, we use simulations to relate Ras three-dimensional orientation at the membrane surface to its signaling competence. Data shows that Ras reorientation is generally rapid, but can be trapped in one of three states by membrane adhesion of the globular signaling domain. One of these states is stabilized by negatively charged lipids and brings an effector binding interface toward the membrane surface, potentially obstructing protein-protein interactions required for propagation of the growth signal. Rare events drive a second type of membrane-based signaling obstruction that correlate with configurational changes in Ras globular domain, yielding a potential drug target.

biophysics↗