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.

Figure 1. Conserved waters in Ras crystal structures, and functionally key regions.

(A) Backbone nitrogen atoms of the Q61H K-ras x-ray structure were used to highlight protein sites occupied by one or more water molecules in >70% (red) and 50–70% (blue) of the available Ras crystal structures (the analyzed structures are listed in Table S1). P-loop (residues 10–17), switch1 (residues 25–40) and switch2 (residues 57–75) are colored in red, green and orange, respectively. The rest of the protein is in yellow cartoon and the molecular surface in transparent light green. (B) Schematic illustration of some of the key residues in the functionally important P-loop and switch regions.

Figure 2. Dynamics of protein-bound waters.

Anomalous behavior of hydration waters in the unrestrained simulations (the F series in Table 1) are characterized by: (A) the radial profile of the diffusion coefficient, D, and (B) the dipole orientation order parameter, <cosθ>, as a function of distance r from the protein surface. Also shown are the dipole autocorrelation function at different r values (C), as well as the survival probability function, N_w(t), of waters in the first hydration shell (D).

Figure 3. A “belt” of long-residence waters in Ras.

(A) Six water molecules (W₁–W₆) found to be stably protein-bound during the unrestrained simulations are shown along with their nearest backbone atoms. The protein is in gray except for the P-loop (red), switch1 (green), switch2 (orange), and helix3 (residues 84–104, yellow). (B) Hydrogen-bonding interactions between backbone or side-chain protein atoms and the six water molecules. Since the interactions involving residues T20, H27, V29 and R68 are dynamic, they are not present in this particular snapshot and hence not shown. Color code: carbon, yellow; oxygen, red; nitrogen, blue.

Figure 4. Analysis of crystal and MD-derived Ras conformers.

(A) Classification of Ras x-ray structures into GTP (red), intermediate (blue), GDP (cyan) and nucleotide-free (green) states based on the distance (d) between the C_α atoms of G10 and G60, and the N-C_α-C-O dihedral of G60 (ξ). PDB ids and exceptions are labeled where space permits. (B) Overlay of MD-derived d/ξ values (light gray) onto those derived from the crystal structures. Also shown is a heat map cluster (red is most intense) of conformers in which W₄ is absent. (C) The time evolution of the water coordination number for G10 carbonyl oxygen. See text for the definition of states.

Figure 5. Re-distribution of conformational states by structural waters.

d/ξ values from MD (gray) are overlaid with those from x-ray structures (symbols) for simulations (A); (B); (C); (D); (E); and (F). See legend of Fig. 4 for color scheme and Table 1 for simulation names.

Figure 6. Superimposition of the average structures of conformers with and without W₅ (red sphere).

Major differences are highlighted in green (with W₅) and yellow (without W₅), showing allosteric modulation of S₂ by W₅ located at the N-terminus of H₃ across the lobe interface.

Figure 7. The role of Y96 in a proposed water-mediated allostery.

(A) Histogram of the Y96:OH-G60:O distance in the unrestrained simulations plotted separately for conformers having d (see Fig. 4) less than (black) and greater than or equal to (red) 7.5 Å. Vertical dashed lines indicate the corresponding values averaged over the GTP- (left) and GDP-bound (right) x-ray structures. (B) Evolution of the angle between vectors connecting C_α atoms of S89 and Y96, and C_γ and OH of Y96 in the concatenated unrestrained simulations. (C) Representative snapshots highlighting conformers in the GTP, GDP and nucleotide-free states.