Sequence alignment of the four WW domains from human Nedd4-1 (neuronal precursor cell expressed developmentally down-regulated gene 4-1) reveals that the highest sequence diversity exists in loop I. Three residues in this type I beta-turn interact with the PPxY motif of the human epithelial Na+ channel (hENaC) subunits, indicating that peptide affinity is defined by the loop I sequence. The third WW domain (WW3*) has the highest ligand affinity and unlike the other three hNedd4-1 WW domains or other WW domains studied contains the highly statistically preferred proline at the (i + 1) position found in beta-turns. In this report, molecular dynamics simulations and experimental characterize loop I stability and dynamics. Exchange of the proline to the equivalent residue in WW4 (Thr) results in the presence of a predominantly open seven residue Omega loop rather than the type I beta-turn conformation for the wild-type apo-WW3*. In the presence of the ligand, the structure of the mutated loop I is locked into a type I beta-turn. Thus, proline in loop I ensures a stable peptide binding-competent beta-turn conformation, indicating that amino acid sequence modulates local flexibility to tune binding preferences and stability of dynamic interaction motifs.