Ras Conformational Switching: Simulating Nucleotide-Dependent Conformational Transitions with Accelerated Molecular Dynamics

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Abstract
Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding Ras signaling. We characterize nucleotide-dependent conformational transition using multiple-barrier-crossing accelerated molecular dynamics (aMD) simulations. These transitions, achieved for the first time for wild-type Ras, are impossible to observe with classical molecular dynamics (cMD) simulations due to the large energetic barrier between end states. Mapping the reaction path onto a conformer plot describing the distribution of the crystallographic structures enabled identification of highly populated intermediate structures. These structures have unique switch orientations (residues 25–40 and 57–75) intermediate between GTP and GDP states, or distinct loop3 (46–49), loop7 (105–110), and α5 C-terminus (159–166) conformations distal from the nucleotide-binding site. In addition, these barrier-crossing trajectories predict novel nucleotide-dependent correlated motions, including correlations of α2 (residues 66–74) with α3-loop7 (93–110), loop2 (26–37) with loop10 (145–151), and loop3 (46–49) with α5 (152–167). The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function. In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras. Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure. The Ras family of enzymes mediate signaling pathways controlling cell proliferation and development by cycling between active and inactive conformational states. Mutations that affect the ability to switch between states are associated with a variety of cancers. However, details of how the structural changes occur and how mutations affect the fidelity of this process remain to be determined. Here we employ an advanced computational technique, termed accelerated molecular dynamics, to characterize structural transitions and identify novel highly populated transient conformations. Several spatially distant structural regions were found to undergo correlated motions, highlighting a dynamic linkage between the sites of enzymatic reaction and the membrane-interacting C-terminus. In addition, our results indicate that the major motion occurring during the conformational exchange is a low-frequency motion intrinsic to the structure. Hence, features of the characterized transitions likely apply to a large number of structurally similar but functionally diverse nucleotide triphosphatases. These results provide fresh insights into how oncogenic mutations might modulate conformational transitions in Ras.