X-ray crystal structure analysis of the catalytic domain of the oncogene product p21H-ras complexed with caged GTP and mant dGppNHp.

The X-ray structures of the 1:1 complexes formed between p21H-ras (residues 1 to 166) and the nucleotides P3-1-(2-nitrophenyl)ethyl guanosine triphosphate ("caged GTP"; pure R- and S-diastereomers) and 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imido)-triphosphate ("mant dG-ppNHp"), have been refined to an R-factor of 21.4% (R-caged GTP, 1.85 A resolution), 18.9% (S-caged GTP, 2.5 A resolution) and 17.6% (mant dGppNHp, 2.7 A resolution), respectively. Details of the structure determination, refinement and the structures themselves are presented. The overall structures of the complexes are identical in terms of the general organization of their secondary structure elements and are also identical to that reported for the analogous complex of p21H-ras with GppNHp. The binding of the GTP part is not significantly affected by the additional aromatic group (cage and mant, respectively) in contrast to the original observation on p21:caged GTP using the racemic mixture of R- and S-caged GTP. The main differences in the structures are observed in the region of loop L2 (residues Glu31 to Thr35) where the additional aromatic group attached to the nucleotide comes very close to the side-chain of Tyr32, including backbone displacements of 2.6 A, 2.2 A and 0.3 A for the residues from Glu31 to Thr35 for R-caged, S-caged GTP and mant dGppNHp, respectively. The refined structures provide additional data for the design of new nucleotide analogs and the importance of their stereochemistry as well as for the design of new mutant forms of p21H-ras for further biochemical investigations. The binding mode of mant dGppNHp reveals significant features for the understanding of the fluorescence signals observed in solution.