The Role of Conserved Waters in Conformational Transitions of Q61H K-ras

Abstract

To investigate the stability and functional role of long-residence water molecules in the Q61H variant of the signaling protein K-ras, we analyzed all available Ras crystal structures and conformers derived from a series of independent explicit solvent molecular dynamics (MD) simulations totaling 1.76 µs. We show that the protein samples a different region of phase space in the presence and absence of several crystallographically conserved and buried water molecules. The dynamics of these waters is coupled with the local as well as the global motions of the protein, in contrast to less buried waters whose exchange with bulk is only loosely coupled with the motion of loops in their vicinity. Aided by two novel reaction coordinates involving the distance ( d) between the C _α atoms of G60 at switch 2 and G10 at the P-loop and the N-C _α-C-O dihedral ( ξ) of G60, we further show that three water molecules located in lobe1, at the interface between the lobes and at lobe2, are involved in the relative motion of residues at the two lobes of Q61H K-ras. Moreover, a d/ξ plot classifies the available Ras x-ray structures and MD-derived K-ras conformers into active GTP-, intermediate GTP-, inactive GDP-bound, and nucleotide-free conformational states. The population of these states and the transition between them is modulated by water-mediated correlated motions involving the functionally critical switch 2, P-loop and helix 3. These results suggest that water molecules act as allosteric ligands to induce a population shift among distinct switch 2 conformations that differ in effector recognition.

Author Summary

K-ras belongs to the Ras family of G-proteins that regulate cell proliferation and development. To execute its function, K-ras adopts different conformational states when it is active and inactive. In addition to these two states, it samples many transient intermediate conformations as it makes the transition from one state to the other. Mutations that affect the population of these states can cause cancer or developmental disorder. Using simulation approaches, here we show that a number of water molecules buried within the structure of an oncogenic K-ras protein modulate the distribution of its conformational states. Moreover, a detailed analysis based on two novel structural parameters revealed the existence of long-range water-mediated interactions that facilitate a dynamic coupling between the two lobes of the protein. These findings pave the way for a dynamics-guided strategy to inhibit abnormal Ras signaling.