Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 314, Issue 3, Pages 577-588Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1006/jmbi.2001.5141
Keywords
FHA domain; Rad53; Rad9; phosphothreonine; NMR structure
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Funding
- NCI NIH HHS [CA87031] Funding Source: Medline
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It was proposed previously that the FHA2 domain of the yeast protein kinase Rad53 has dual specificity toward pY and pT peptides. The consensus sequences of pY peptides for binding to FHA2, as well as the solution structures of free FHA2 and FHA2 complex with a pY peptide derived from Rad9, have been obtained previously. We now report the use of a pT library to screen for binding of pT peptides with the FHA2 domain. The results show that FHA2 binds favorably to pT peptides with Ile at the +3 position. We then searched the Rad9 sequences with a pTXXI/L motif, and tested the binding affinity of FHA2 toward ten pT peptides derived from Rad9. One of the peptides, (EVEL)-E-599(pT)QELP(607), displayed the best binding affinity (K-d = 12.9 muM) and the greatest chemical shift changes. The structure of the FHA2 complex with this peptide was then determined by solution NMR and the structure of the complex between FHA2 and the pY peptide (EDI)-E-826(pY)YLD832 was further refined. Structural comparison of these two complexes indicates that the Leu. residue at the +3 position in the pT peptide and that at the +2 position in the pY peptide occupy a very similar position relative to the binding site residues from FHA2. This can explain why FHA2 is able to bind both pT and pY peptides. This position change from +3 to +2 could be the consequence of the size difference between Thr and Tyr. Further insight into the structural basis of ligand specificity of FHA domains was obtained by comparing the structures of the FHA2-pTXXL complex obtained in this work and the FHA1-pTXXD complex reported in the accompanying paper. (C) 2001 Academic Press.
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