Solution structure of the yeast Rad53 FHA2 complexed with a phosphothreonine peptide pTXXL: Comparison with the structures of FHA2-pYXL and FHA1-pTXXD complexes

It was proposed previously that the FHA2 domain of the yeast protein kinase Rad53 has dual specificity toward pY and pT peptides. The consensus sequen ces 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 bee n 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 bin ds favorably to pT peptides with Ile at the +3 position. We then searched t he 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) an d the greatest chemical shift changes. The structure of the FHA2 complex wi th this peptide was then determined by solution NMR and the structure of th e complex between FHA2 and the pY peptide (EDI)-E-826(pY)YLD832 was further refined. Structural comparison of these two complexes indicates that the L eu. residue at the +3 position in the pT peptide and that at the +2 positio n 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 consequenc e of the size difference between Thr and Tyr. Further insight into the stru ctural basis of ligand specificity of FHA domains was obtained by comparing the structures of the FHA2-pTXXL complex obtained in this work and the FHA 1-pTXXD complex reported in the accompanying paper. (C) 2001 Academic Press .