WW domains are universal protein modules for binding Pro-rich ligands. They are classified into four groups according to their binding specificity. Arg-14 and Arg-17, on the WW domain of Pin1, are thought to be important for the binding of Group IV ligands that have (Ser(P)/Thr(P))-Pro sequences. We have applied surface plasmon resonance to determine the ligand specificity of several WW domains containing Arg-14. Among these WW domains, Rsp5.2 and mNedd4.3 bound only to the Group I ligand containing Pro-Pro-Xaa-Tyr with K D values of 11 and 55 M, respectively. The WW domains of hPin1, Caenorhabditis elegans Pin1 homologue (Y110), PinA, and SspI bound to Group IV ligands with K D values ranging from 22 to 700 M. PinA and SspI do not have Arg-17, unlike Pin1 and Y110. The modeled structures of the WW domains of PinA and SspI revealed that the structure and the network of hydrogen bonds of Loop I, which are also formed in Pin1 and Y110, are conserved. We propose that this configuration of Loop I (referred to as the "p patch") is necessary for binding Group IV ligands and that it can be used to predict the specificity and functions of other WW domains.A WW domain is a small structural motif named after a pair of tryptophan residues that are highly conserved (1, 2). It works as an interaction module in proteins with various functions, including cell cycle control (Pin1/Ess1), ubiquitin ligation (Nedd4/Rsp5 and Smurf1), and coactivation of transcription (YAP65) (3, 4). Some WW domain-containing proteins are related directly or indirectly to a variety of human diseases such as Liddle's syndrome, Duchenne muscular dystrophy, Huntington's disease, and Alzheimer's disease (3,5,6).WW domains bind proline-containing peptide sequences and can be classified into four groups according to their ligand specificity: Group I domains recognize Pro-Pro-Xaa-Tyr (PPXY) (2); Group II, Pro-Pro-Leu-Pro; Group III, Pro-rich sequences with Arg; and Group IV, (Ser(P)/Thr(P))-Pro (1). Human Pin1 (hPin1) and its yeast homologue, ESS1, are parvulin-type PPIases connected to Group IV WW domains. They are essential regulators for mitotic arrest (7,8). hPin1 binds to mitotically phosphorylated proteins such as Cdc25c, Myt1, Plk1, and Cdc27 through its WW domain (7), and it also binds to the phosphorylated RNA pol II 1 C-terminal domain (CTD), implying transcriptional regulation by Pin1 (3).Several structures have been determined so far for WW domain-ligand complexes, three of Group I and one of Group IV. These structures reveal that WW domains each adopt a triple-stranded antiparallel -sheet. For each domain, the same side of the -sheet is used to bind all of its respective ligands (2, 6, 9, 10). The crystal structure of the hPin1 WW domain, complexed with a phosphorylated CTD peptide of RNA polymerase II (Tyr-Ser(P)-Pro-Thr-Ser(P)-Pro-Ser), has shown the importance of two arginine residues (Arg-14 and Arg-17) on this side of the sheet in recognizing the ligand. Arg-14 of the WW domain recognizes Pro-3Ј of the ligand peptide, whereas Arg-17 int...