Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP.

Four overlapping peptide fragments of human c-Raf-1 (residues 55-132, 55-117, 77-132, and 77-117) were expressed in Escherichia coli as carboxyl-terminal extensions of maltose binding protein (MBP). The MBP-Raf fusions were purified by affinity chromatography on amylose resin and tested for binding to Ras.GTP indirectly by measuring their ability to inhibit the stimulation of Ras GTPase activity by GTPase activating protein (GAP120) in vitro. MBP-Raf(55-132) was a potent inhibitor in this assay (50% inhibition at 100 nM concentration), but the other fusion proteins had no measurable effect. The fusion partners were cleaved with Factor Xa protease and separated by gel filtration. The 8960-dalton Raf(55-132) fragment retained full activity as a competitive inhibitor of GAP120. It also blocked Ras-stimulated germinal vesicle breakdown in frog oocytes. Raf(55-132) was further characterized by circular dichroism and nuclear magnetic resonance spectroscopy. The results indicate that this fragment of c-Raf-1 adopts a highly structured, monomeric conformation in solution.

Chemical shift assignments and folding topology of the Ras-binding domain of human Raf-1 as determined by heteronuclear three-dimensional NMR spectroscopy.

Raf-1 is a 74-kDa serine-threonine kinase which serves as the immediate downstream target of Ras in the cell growth signal transduction pathway. Recent genetic and biochemical experiments have demonstrated that (1) Ras interacts directly with the amino-terminal domain of Raf and (2) residues 51-131 of the Raf sequence are sufficient to mediate this interaction [Vojtek, A. B., Hollenberg, S. M., & Cooper, J. A. (1993) Cell 74, 205-214]. We have expressed a corresponding segment of the human Raf sequence (Raf55-132) in Escherichia coli as a fusion with maltose binding protein. The fusion protein was purified by affinity chromatography and cleaved at a pre-engineered site with factor Xa protease to liberate the 78-residue fragment of Raf. Raf55-132 bound to Ras with high affinity in a competition assay with GAP. An unlabeled version of Raf55-132 was studied by 2D homonuclear NMR, and uniformly 15N- and 13C/15N-labeled versions of Raf55-132 were studied by 2D and 3D heteronuclear NMR. Nearly complete sequence-specific assignments were made for the backbone HN, H alpha, 15N, and 13C alpha resonances. NOEs were used to determine regions of secondary structure and the overall folding topology. Raf55-132 is an independently folded domain composed of a five-stranded beta-sheet, a three-turn alpha-helix, and possibly an additional one-turn helix. Its structure resembles that of ubiquitin, even though there is no more than 11% sequence homology between the two proteins.