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Solution structure of the Ras binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe
W Gronwald1, F Huber, P Grünewald
1Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Postfach, D-93040, Regensburg, Germany.
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.
Insights
The 3D structure of fission yeast Byr2 Ras binding domain (Byr2RBD) reveals a ubiquitin superfold. Despite low sequence identity, Byr2RBD shares structural motifs with other Ras binding domains, but with distinct interaction interfaces for specific Ras recognition.
Area of Science:
- Structural Biology
- Molecular Biology
- Biochemistry
Background:
- Small GTPases like Ras signal to effectors via specific binding domains.
- Effector binding domain structures dictate downstream pathway activation.
- Byr2 (fission yeast) is a Raf homolog, crucial for Ras signaling, but its 3D structure and Ras binding domain sequence similarity to Raf are poorly understood.
Purpose of the Study:
- To determine the three-dimensional structure of the Ras binding domain of Byr2 (Byr2RBD) from Schizosaccharomyces pombe.
- To investigate the interaction between Byr2RBD and Ras using biophysical methods.
- To elucidate the structural basis for Ras recognition by Byr2.
Main Methods:
- Solution Nuclear Magnetic Resonance (NMR) spectroscopy, specifically 15N-(1)H-TROSY-HSQC.
- 3D structure determination of Byr2RBD.
- Analysis of Byr2RBD complex with Ras*Mg(2+)*GppNHp.
Main Results:
- The 3D structure of Byr2RBD was solved, revealing three alpha helices and a five-stranded beta sheet with a ubiquitin superfold topology.
- NMR studies showed that the first and second beta strands and the first alpha helix of Byr2RBD are involved in Ras binding.
- Differences in NMR spectra upon binding of S. pombe Ras1 to Byr2RBD, compared to human H-Ras, indicate distinct binding modes despite sequence identity at the interaction site.
Conclusions:
- The ubiquitin superfold is a conserved structural motif for Ras binding domains, even with minimal sequence homology.
- Structural variations in the 3D structure and interaction interfaces of Ras binding domains confer specificity in recognizing Ras and related proteins.