Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP.

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.

The locked rotation function.

It frequently occurs that a biological assembly in a crystallographic asymmetric unit has more than one noncrystallographic symmetry operator. For instance, a tetramer might have the point group 222 or a spherical virus will have the point group 532. A self-rotation function searches for the direction and angle of rotation of the individual noncrystallographic symmetry operations, while a cross-rotation function searches for the relationship of a structure in one unit cell with similar structures in another cell. The power of the rotation function can be greatly enhanced by searching for all noncrystallographic symmetry operators simultaneously. The procedure described previously [Rossmann, Ford, Watson & Banaszak (1972). J. Mol. Biol. 64, 237-249] has been generalized. The increased power of this 'locked' rotation function permits a good determination of the orientation of an icosahedral virus in the presence of less than 1% of the possible diffraction data to 7 A resolution. In addition, the peak-to-noise ratio is substantially improved.

Structural differences between a ras oncogene protein and the normal protein.

One of the most commonly found transforming ras oncogenes in human tumours has a valine codon replacing the glycine codon at position 12 of the normal c-Ha-ras gene. To understand the structural reasons behind cell transformation arising from this single amino acid substitution, we have determined the crystal structure of the GDP-bound form of the mutant protein, p21(Val-12), encoded by this oncogene. We report here the overall structure of p21(Val-12) at 2.2 A resolution and compare it with the structure of the normal c-Ha-ras protein. One of the major differences is that the loop of the transforming ras protein that binds the beta-phosphate of the guanine nucleotide is enlarged. Such a change in the 'catalytic site' conformation could explain the reduced GTPase activity of the mutant, which keeps the protein in the GTP bound 'signal on' state for a prolonged period time, ultimately causing cell transformation.