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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.
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