Structural Studies of SH3 and SH2 Domain of Bruton's Tyrosine Kinase
2001
Hochschulschrift
Zugriff:
89
X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton''s tyrosine kinase (BTK). Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in a patient family. We studied binding of BTK SH3 domain (aa 216—273, 58 residues) and truncated SH3 domain (aa 216—259, 44 residues) with proline-rich peptides. We show that SH3 and truncated SH3 domains bind to BTK TH domain peptide and peptide of p120cbl, but truncated SH3 domain binds weakly. This suggests that BTK SH3 and TH domains may associate in inter- or intramolecular fashion, which raises the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand binding modules. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context, hence the weaker binding. Therefore, we have determined the solution structure of BTK SH3 domain and also its complex with a proline-rich peptide from the protein product of c-cbl protooncogene (p120cbl). Like other SH3 domains, BTK SH3 domain consists of five b-strands packed in two b-sheets forming a b-barrel like structure and the proline-rich peptide from p120cbl binds to BTK SH3 domain in a class I orientation. The rmsd for BTK SH3 domain structures, calculated from their averaged coordinates was 0.67 Å (± 0.16 Å) for the backbone heavy atoms (N, C, and Ca) and 1.27 Å (± 0.18 Å) for all heavy atoms. For the b-sheets alone, the rmsd of the 20 structures was 0.24 Å (± 0.06 Å) for backbone heavy atoms and 1.03 Å (± 0.21 Å) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between b4 and b5 of BTK SH3 domain are involved in ligand binding. Furthermore, we have determined the stability and peptide binding affinity of the BTKSH2 domain. Our results indicate that both the structure and stability of BTK SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and I370M mutants of BTK SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200 fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and I370 mutations in the pY+3 hydrophobic binding pocket (~3 to 17 folds). Mutation of the Tyr residue at the bD5 position reverses the binding order of BTK SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. These results delineate the importance of C-terminal in binding of SH3 domains and indicate also that improper folding and the altered binding behavior of mutant BTK SH3 or SH2 domain likely leads to XLA.
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Structural Studies of SH3 and SH2 Domain of Bruton's Tyrosine Kinase
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Autor/in / Beteiligte Person: | TZENG, SHIOU-RU ; 曾秀如 |
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Veröffentlichung: | 2001 |
Medientyp: | Hochschulschrift |
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