H-1 NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides

The recent crystal structure of Pin1 protein bound to a doubly phosphorylat ed peptide from the C-terminal domain of RNA polymerase II revealed that bi nding interactions between Pin1 and its substrate take place through its Tr p-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro pe ptide bonds. However, the orientation of the ligand in the aromatic recogni tion groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A, and Lim, W, A (2000) Nat, Struct, Riot, 7, 611-613), Because the bound peptide conformation could also differ as observed for peptide l igands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here s tudied by solution NMR methods. First, the proton resonance perturbations o n the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta -hairpin Ser(11)- Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 muM for Cdc25 and tau peptides, respectively, were found. S everal intermolecular nuclear Overhauser effects between WW domain and subs trates were obtained from a ligand-saturated solution and were used to dete rmine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide lig ands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.