Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope

The single mutation L30 K in the Hu-Yap65 WW domain increased the stability of the complex with the peptide GTPPPPYTVG (Kd = 40(±5) μM). Here we report the refined solution structure of this complex by NMR spectroscopy and further derived structure-activity relationships by using ligand peptide libraries with truncated sequences and a substituion analysis that yielded acetyl-PPPPY as the smallest high-affinity binding peptide (Kd = 60 μM). The structures of two new complexes with weaker binding ligands chosen based on these results (N-(n-octyl)-GPPPYNH2 and Ac-PLPPY) comprising the wild-type WW domain of Hu-Yap65 were determined. Comparison of the structures of the three complexes were useful for identifying the molecular basis of high-affinity: hydrophobic and specific interactions between the side-chains of Y28 and W39 and P5′ and P4′, respectively, and hydrogen bonds between T37 (donnor) and P5′ (acceptor) and between W39 (donnor) and T2′ (acceptor) stabilize the complex.