Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf

Abstract

Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the gamma-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.

Figure 1

Ribbon diagram of Ras in the R32 crystal form. Two molecules are shown to illustrate crystal contacts along the 2-fold symmetry axes involving switch I. Switch II residues 61−68 are removed from the model. Residues 59−60 and 69−72 are shown in red. The GTP analog, GppNHp, as well as Ca²⁺ ions are yellow. The Raps/RafRBD complex (PDB code 1GUA) is shown in gray, superimposed on the Ras molecule in green based on the nucleotide. Ras (Raps) residues 31−33 and Raf Lys84 are in stick. Figures 1-5 were generated with PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA).

Figure 2

Electron density for switch II residues 61−70. (A) RasQ61L-GppNHp. (B) RasQ6V-GppNHp. (C) RasQ61K-GppNHp (D) RasQ61I-GppNHp (R32). All panels show final 2F_o-F_c electron density maps contoured at the 1σ level.

Figure 3

Superposition of transforming Ras-GppNHp Q61 mutants (residues 32−35 and 61−64) and Ran-GppNHp/importinβ (residues 40−43 and 69−72). GppNHp is in yellow. The mutant structures are colored as follows: Q61L, cyan; Q61K, green; Q61I, magenta; Q61V, orange. Ran is in light gray. Wat175 corresponds to the pre-catalytic water molecule. Red dashed lines represent hydrogen bonds.

Figure 4

Active site surfaces in the RasQ61 mutants and in the wild type structure in complex with GppNHp (yellow). (A) Q61L; (B) Q61V; (C) Q61K; (D) Q61I (R32), (E) Q61G (PDB entry 1ZW6); (F) Wild Type (R32). Surfaces were constructed using PyMOL and colored based on atom type (N, blue; O, red; C, green). Only protein atoms were used to define the surface. Areas outlined in white define the protein/solvent interface. Wat175 represents the pre-catalytic water molecule.

Figure 5

Switch I in the wild type and mutant Ras structures: comparisons with biologically relevant complexes. (A) Wild type Ras-GppNHp (green with water molecules in red) and Raps-GppNHp/Raf-RBD (yellow with water molecules in orange). (B) RasQ61L-GppNHp (green with water molecules in red) and Ran-GppNHp-Importinβ (yellow with water molecules in orange). The nucleotide is in gray. Red dashed lines represent hydrogen bonds.

Figure 6

Gel filtration chromatography results from the hydrolysis experiments. A) RasQ61L incubated with Raf. The contents of the elution peaks are shown in the inserted SDS gel. Lane 1: MW markers; Lane 2: sample injected into the column; Lanes 3 and 4: Raf, which tends to aggregate at 4°C and elute near the void volume (not shown in elution profile); Lanes 5 and 6: fractions taken from the first elution peak showing a 1:1 ratio of Raf and Ras; Lanes 7, 8 and 9: second elution peak containing the Ras protein. B) RasQ61L incubated without Raf. C) Wild type Ras incubated with Raf. D) Wild type Ras incubated without Raf. For results shown in B and D Raf was added before gel filtration.