The Distinct Conformational Dynamics of K-Ras and H-Ras A59G

Abstract

Ras proteins regulate signaling cascades crucial for cell proliferation and differentiation by switching between GTP- and GDP-bound conformations. Distinct Ras isoforms have unique physiological functions with individual isoforms associated with different cancers and developmental diseases. Given the small structural differences among isoforms and mutants, it is currently unclear how these functional differences and aberrant properties arise. Here we investigate whether the subtle differences among isoforms and mutants are associated with detectable dynamical differences. Extensive molecular dynamics simulations reveal that wild-type K-Ras and mutant H-Ras A59G are intrinsically more dynamic than wild-type H-Ras. The crucial switch 1 and switch 2 regions along with loop 3, helix 3, and loop 7 contribute to this enhanced flexibility. Removing the gamma-phosphate of the bound GTP from the structure of A59G led to a spontaneous GTP-to-GDP conformational transition in a 20-ns unbiased simulation. The switch 1 and 2 regions exhibit enhanced flexibility and correlated motion when compared to non-transitioning wild-type H-Ras over a similar timeframe. Correlated motions between loop 3 and helix 5 of wild-type H-Ras are absent in the mutant A59G reflecting the enhanced dynamics of the loop 3 region. Taken together with earlier findings, these results suggest the existence of a lower energetic barrier between GTP and GDP states of the mutant. Molecular dynamics simulations combined with principal component analysis of available Ras crystallographic structures can be used to discriminate ligand- and sequence-based dynamic perturbations with potential functional implications. Furthermore, the identification of specific conformations associated with distinct Ras isoforms and mutants provides useful information for efforts that attempt to selectively interfere with the aberrant functions of these species.

Author Summary

The proto-oncogene Ras mediates 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 over 25% of human tumors. However, despite much effort, details of how these mutations affect the fidelity of activating conformational transitions remain unclear. Here we employ extensive molecular dynamics simulations combined with principal component analysis to investigate whether the subtle differences among functionally distinct isoforms and oncogenic mutants are associated with detectable dynamical differences. Our results reveal that wild-type K-Ras, the most prevalent isoform in a number of cancers, and mutant H-Ras A59G are intrinsically more dynamic than wild-type H-Ras. Furthermore, we have observed the first spontaneous GTP-to-GDP transition of H-Ras A59G during unbiased molecular dynamics simulation. These results indicate that key changes in sequence can lead to different dynamic properties that may be relevant for the unique physiological and aberrant functions of Ras isoforms and mutants. Furthermore, the current results shed further light on the conformational transition mechanism of this important molecular switch.