Solution structure of the Ras binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe.
BACKGROUND
After activation, small GTPases such as Ras transfer the incoming signal to effectors by specifically interacting with the binding domain of these proteins. Structural details of the binding domain of different effectors determine which pathway is predominantly activated. Byr2 from fission yeast is a functional homolog of Raf, which is the direct downstream target of Ras in mammalians that initiates a protein kinase cascade. The amino acid sequence of Byr2's Ras binding domain is only weakly related to that of Raf, and Byr2's three-dimensional structure is unknown.


RESULTS
We have solved the 3D structure of the Ras binding domain of Byr2 (Byr2RBD) from Schizosaccharomyces pombe in solution. The structure consists of three alpha helices and a mixed five-stranded beta pleated sheet arranged in the topology betabetaalphabetabetaalphabetaalpha with the first seven canonic secondary structure elements forming a ubiquitin superfold. 15N-(1)H-TROSY-HSQC spectroscopy of the complex of Byr2RBD with Ras*Mg(2+)*GppNHp reveals that the first and second beta strands and the first alpha helix of Byr2 are mainly involved in the protein-protein interaction as observed in other Ras binding domains. Although the putative interaction site of H-Ras from human and Ras1 from S. pombe are identical in sequence, binding to Byr2 leads to small but significant differences in the NMR spectra, indicating a slightly different binding mode.


CONCLUSIONS
The ubiquitin superfold appears to be the general structural motif for Ras binding domains even in cases with vanishing sequence identity. However, details of the 3D structure and the interacting interface are different, thereby determining the specifity of the recognition of Ras and Ras-related proteins.
Gronwald
W
W
Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Postfach, D-93040, Regensburg, Germany.
Huber
F
F
Grünewald
P
P
Spörner
M
M
Wohlgemuth
S
S
Herrmann
C
C
Kalbitzer
H R
HR
D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry; D020930:MAP Kinase Kinase Kinases; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
D005656:Fungal Proteins; D029702:Schizosaccharomyces pombe Proteins; D006165:Guanylyl Imidodiphosphate; D020928:Mitogen-Activated Protein Kinases; C077892:BYR2 protein, S pombe; D020930:MAP Kinase Kinase Kinases; D018631:ras Proteins