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.
Emerson
S D
SD
Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110.
Waugh
D S
DS
Scheffler
J E
JE
Tsao
K L
KL
Prinzo
K M
KM
Fry
D C
DC
D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014452:Ubiquitins / Q000737:chemistry
D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)