Ludwig Inst. for Cancer Research, Parkville, Vic. 3050, Australia.
Ras is a monomeric GTP dependent signal transducer. It plays an essential role in signal transduction pathways for cellular growth, differentiation and transformation. The active GTP bound Ras can binding to the N-terminal region of the Ser/Thr kinase Raf. The secondary structure structure of the Ras-binding domain of Raf (residues 55-132) has been shown to be homologous to ubiquitin, a functionally unrelated protein (Emerson et al., 1994). The three-dimensional structure of ubiquitin has been solved by X-ray crystallography (Vijay-Kumar et al., 1987) and NMR (Weber et al., 1987; DiStefano & Wand, 1987) .
The secondary structure of Raf55-132 and the [[beta]]-sheet topology has been assigned by NMR (Emerson et al., 1994). Based on this knowledge and the structural similarity between ubiquitin and Raf55-132, a homology model of Raf55-132 was created. Because of a low similarity between the amino acid sequences between the two proteins, we employed a new homology modelling method which is based on conserved distances between hydrophobic amino acids and the conserved overall fold. A simulated annealing protocol is used to create families of structures similar to procedures used for NMR protein structure re<<nement. The procedure starts from randomized sets of atomic coordinates and allows the creation of a number of alternate trial 3D structures for the unknown protein sequence. The trial structures are evaluated using experimental data and well known threading algorithms The most successful structures are re<<ned by an extensive simulated annealing procedure.
Based on known interaction sites between Raf55-132 and Ras we also created a model of the Raf/Ras complex.
Our models of Raf and the Raf/Ras complex in conjunction with site-directed mutagenesis are used to explore the possibility to design new molecules which can block the Raf/Ras interaction.
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